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Number and organization of actin-Related sequences in the mouse genome
Abstract
Recombinant plasmids containing cDNA sequences complementary to the two mouse striated-muscle actin messenger RNAs (pAF81, pAM91) and to a non-muscle actin mRNA (pAL41) have been used to examine the number and organization of actin-related sequences in the mouse genome. A large number (greater than 20) of actin-related sequences are detected on Southern blots of restricted mouse DNA, the majority of which hybridize to both the 5' and 3' ends of the actin-coding sequence, even under conditions revealing only sequences greater than 80% homologous to the actin cDNA probes. More stringent washing of these blots indicates that the two striated muscle actins are each encoded by single genes, and that a non-muscle (beta or gamma) actin cDNA detects one homologous and two closely related sequences in mouse DNA. The segregation of the two striated-muscle actin genes in recombinant inbred mouse strains shows that these genes are not closely linked (greater than 1 centimorgan), and that the skeletal muscle actin gene is not linked to a non-muscle actin gene. Screening a bank of mouse genomic DNA, cloned in Charon 4A, indicates that the number of actin-related sequences in the mouse genome is much higher than 20. In particular, five phages have been isolated representing part of a sub-family of 20 to 50 similar but non-identical sequences, only weakly homologous to actin cDNA probes (probably a family of actin pseudogenes), which are the result of a recent amplification of a greater than 17 X 10(3) base region of mouse DNA.
... Multiple actin isoforms have been identified (6,14,16,47), and some of these proteins are associated with diverse functions such as muscle contraction, cytoskeletal structure, cell motility, and chromosome movement (33). Recombinant DNA techniques have shown that actin isoforms are represented by multigene families in a broad host of eucaryotic genomes (6,13,23,30,32,38,40) except that of Saccharomyces cerevisiae (15). ...
... A major portion of mammalian actin genes encode the cytoplasmic ,B and y actins. In the human genome, P actin alone is represented by at least eight different genes, many of which have been found to be pseudogenes (10,30). Recombination of actin genes with other structural genes might partially explain the complexity of mammalian actin gene families. ...
... In addition, gene duplication events have also contributed to the complexity of mammalian actin genes. Minty et al. (30) showed that a subgroup of nonmuscle actin genes was amplified and integrated into the mouse genome. In contrast to the cytoplasmic actins, the striated muscle actin isoforms are represented in both avian and mammalian genomes as single-copy genes (34). ...
... The following eDNA probes were labeled by nick translation (Rigby et al., 1977) using [s2P]dATP and [32p]dCTP (400 Ci/mmol; Amersham Corp., Arlington Heights, IL) to a specific activity of ,M0 s cpm/#g DNA: pMCK-4 (rat skeletal MCK; Benfield et al., 1984); pAC269 (chicken skeletal muscle ot-actin; Schwartz et al., 1980); pT1 (ot-tubulin; Valenzuela et al., 1981); rat fast skeletal muscle eDNA clones (Garfinkel et al., 1982); pTnT15 (TnT); pMLC-84 (MLC 1); pMLC2-18 (MLC 2); a eDNA clone encoding rat striated muscle a-Tin (Wieczorek et al., 1988); a clone encoding a myosin regulatory light chain (RLC) isolated from a rat aortic smooth muscle cell eDNA library (Taubman et al., 1987); pMHC25 (rat embryonic MHC; Medford et al., 1980); and SMHC 29 (rabbit smooth muscle MHC; Nagai et al., 1988). Synthetic oligonucleotide probes comprised of the first 87 nucleotides of the 3' untransla~l sequences of mouse skeletal a-actin (Hu et al., 1986) and the first 55 nucleotides of the 5' untranslated sequences of mouse cardiac ot-actin (Minty et al., 1983) were end-labeled with T4 polynucleotide kinase and [s2P-3,]ATP. ...
... It has recently been reported that induction of BC3H1 cells results not only in the synthesis of vascular smooth muscle ct-actin, but of skeletal a-actin as well (Reeser, J. C., and A. R. Strauch, unpublished results). RNA from BC3H1 cells was hybridized to probes specific for the 5' untranslated end of mouse cardiac t~-actin (Minty et al., 1983) and 3' untranslated end of mouse skeletal ot-actin (Hu et al., 1986) that seen in rat aomc smooth muscle cells, where increasing amounts of RLC mRNA are detected at confluence (Taubman et al., 1987). Thus, BC3H1 cells express both sarcomeric and nonsarcomeric forms of myosin RLC, with the former present predominantly in the growth-arrested, differentiated state and the latter predominantly during rapid cell growth. ...
The BC3H1 cell line has been used widely as a model for studying regulation of muscle-related proteins, such as the acetylcholine receptor, myokinase, creatine kinase, and actin. These cells, derived from a nitrosourea-induced mouse brain neoplasm, have some of the morphological characteristics of smooth muscle and have been shown to express the vascular smooth muscle isoform of alpha-actin. To provide further information about the contractile protein phenotype of BC3H1 and to gain additional insights into the possible tissue of origin of these cells, we have examined the expression of a battery of contractile protein genes. During rapid growth, subconfluent BC3H1 cells express the nonmuscle isoform of alpha-tropomyosin (alpha-Tm) and the nonsarcomeric isoforms of myosin heavy and light chains (MHCs and MLCs, respectively), but do not express troponin T(TnT). However, when BC3H1 cells differentiate in response to incubation in serum-deprived medium or upon approaching confluence, they express TnT as well as sarcomeric muscle isoforms of MHC, MLC 2 and 3, alpha-Tm, and alpha-actin. These results suggest that BC3H1 is a skeletal muscle cell line of ectodermal origin that is defective for commitment to terminal differentiation.
... The genomes of yeast, protists and metazoans contain from 1 to 17 actin genes (see Table 1). Actin multigene families in mammals are complex and their members are generally not linked [7,14,20]. The human genome contains genes encoding six actin isoforms and more than thirty pseudogenes with sequences homologous to the coding sequences of beta and g a m m a cytoplasmic actins [25,26]. ...
... The murine genome also contains genes which encode 6 actin isoforms plus 20 to 50 D N A sequences which are weakly homologous to actin cDNA probes. These are probably a family of actin pseudogenes which arose from a recent amplification of greater than 17 kb of the murine genome [20]. Within species, plant actins are more divergent than those of any other group of or- ...
The actin gene superfamily of Petunia hybrida cv. Mitchell contains greater than 100 gene members which have been divided into several highly divergent subfamilies [1]. Five subfamily-specific probes have been used to compare the actin genes among the Mitchell, Violet 23 (V23) and Red 51 (R51) cultivars of P. hybrida. The sum total of actin genes in these five subfamilies was estimated to be between 10 and 34 members in both V23 and R51. Restriction fragment length polymorphisms (RFLPs) between V23 and R51 were examined with these five probes and eleven different restriction endonucleases. Among the 55 comparisons, 87% exhibited RFLPs. These data indicate extreme divergence between V23 and R51 in DNA sequence and/or the presence of small insertions and deletions surrounding these actin gene subfamilies. This divergence suggests that V23 and R51, which have contrasting phenotypic marker loci on every chromosome, may be useful for the development of a complete RFLP linkage map of the Petunia genome. The segregation of Hind III RFLPs among the progeny of two backcrosses demonstrated that representatives of the five subfamilies of Petunia actin genes exist at four distinct genetic locations and suggested that two of these loci are tightly linked. Apparently, amplification of the numerous members of the Petunia actin gene superfamily occurred via gene dispersal of the original subfamily progenitors and not primarily as a result of amplification of a single chromosomal region.
... One of the first subjects to be addressed with the newly cloned actin cDNAs, was the number of actin genes in mammalian genomes. We found that in mouse DNA, in addition to the six actin genes expected from protein sequencing work, there was also a family of weakly-related sequences, some of which had recently been amplified in the genome [11]. Although these had not been sequenced, the length of these actin-like genes in genomic DNA led us to predict that they could represent intron-less pseudogenes, generated by reverse transcription of actin mRNAs and insertion in the genome. ...
Although reflection is obviously crucial in molecular biology, experimentation is nonetheless the basis of most major advances. I was lucky to begin my research career at a particularly interesting time, and privileged to have spent a number of years in Francois Gros' laboratory at the Institut Pasteur. His influence, and that of his lab, were crucial in shaping my early career.
... NaCl, 0.15 M sodium citrate, pH 7). The membranes were hybridized with the 32 P-labelled 0.5 kb cDNA of the murine EpoR purified from Puc 18 plasmid (kindly provided by Catherine Lacombe MD, INSERM Unit 152, Paris) [17], and the 32 P-labelled 1.1 kb cDNA of the mouse p actin gene [18]. ...
Background
The treatment of uraemic patients with recombinant human erythropoietin (rHuEpo) often leads to an increase in blood pressure. Indirect and direct effects of the hormone are probably involved. We explored the possibility of a direct action on the vascular smooth muscle cell (VSMC).
Methods
Rat VSMC were isolated from aortas of spontaneously hypertensive rats (SHR) and normotensive control rats (WKY) and maintained in culture. They were exposed to rHuEpo under various experi mental conditions, and cells proliferative index was measured by [³H]-thymidine incorporation. Binding studies and Northern blots were performed in an attempt to identify a specific erythropoietin receptor (EpoR). In the latter experiment, Epo-responsive Rauscher Reds cells (Reds cells) were used as a positive control for mRNA EpoR expression.
Results
VSMC growth index of SHR was enhanced up to 1.6-fold by rHuEpo concentrations of 16 U/ml or more, in the presence of 1% fetal calf serum. No such stimulation was observed in VSMC of WKY. Binding studies with radiolabelled rHuEpo showed either extremely low or no specific binding of radio-labelled rHuEpo by VSMC. However, Northern blot analysis revealed the expression of EpoR mRNA in VSMC of either rat strain.
Conclusion
The present report provides preliminary evidence in favour of a direct action of the hormone on vascular smooth muscle via a specific EpoR.
... Probes were either the Y-terminal 1 kb EcoRV-Hindlll fragment, the central 3 kb Hindlll-Hindlll fragment, or the 3'-terminal 1 kb Hindlll-BamHl fragment of MR cDNA. For controls, Iwe used the 1.0 kb EcoRI-Hindlll 5'.terminal fragment of mouse transferrin receptor cDNA clone pTfR2 (Stearne et al., 1985), the 0.4 kb Smal-Pstl fragment of human HLA-A2 gene encompassing exon 3 (Keller and Orr, 1985), the 0.3 kb Hhal-EcoRI fragment of mouse !32-microglobulin cDNA clone pB2-m2 (positions 23-317) (Daniel et al., 1983), or the 1.1 kb Pstl-Pstl fragment of mouse B-actin cDNA clone pAL41 (Minty et al., 1983). After hybridization, the filters were washed with 0.1x SSC, 1% SDS, at 60°C to avoid cross-reaction of the MA probes with endogenous mouse sequences. ...
... The probes used were the 1,000-bp Pst 1 insert of human PAIR cDNA (17) (kindly provided by Dr. David Loskutoff, Scripps Clinic and Research Foundation, La Jolla, CA) ; the Eco RI insert of pPAI J7, harboring 1,900 by of the human PAI-2 cDNA (18) ; the Bgl II fragment of pPA 11 4B cDNA harboring 1,948 by of the human t-PA cDNA (19) ; the Eco Rl fragment of pUK 0321 harboring 1,023 by of the human u-PA cDNA (15) . Mouse 0-actin cDNA (20) was a generous gift from Dr. Stefania Petrucco, Swiss Institute for Cancer Research, Epalinges, Switzerland ; a human genomic PAI-2 clone (chPAI-2 8) was isolated from a human Charon 4A gene library using the labeled Eco RI insert of pPAI J7 (above) . The authenticity of the clone was established by sequencing the first two exons . ...
Human rTNF/Cachectin was shown to stimulate gene transcription of plasminogen activator inhibitor (PA1)-1 and PAI-2, and simultaneously suppress constitutive gene expression of tissue-type plasminogen activator (t-PA) in human fibrosarcoma cells. We propose that a TNF-mediated reprogramming of gene transcription induces, in appropriate target cells, an anti-fibrinolytic state, which may cooperate with the induction of procoagulant activity (tissue factor) to stabilize the fibrin deposits commonly found in inflamed tissue. PAI genes also provide a model system for a study of the molecular pathways underlying TNF-mediated signal transduction.
... This additional band cannot be explained by the DNAs from nit1/cw15 (n) and ida5-t (t) were digested with a restriction enzyme shown at the bottom and probed with the probes indicated in the restriction map (Sugase et al., 1996) known actin sequence and may well have originated from the gene of NAP. Whatever the gene is, it may not be highly homologous to conventional actin, because it cannot be detected at higher stringencies; if the two genes were members of a gene family of conventional actin, like the multiple actin genes in other organisms such as Dictyostelium (Romans and Firtel, 1985) and mouse (Minty et al., 1983), the two sets of bands might well have appeared even under high stringency conditions. It is likely that conventional actin is totally missing in the ida5 mutants. ...
Chlamydomonas flagellar inner-arm dynein consists of seven subspecies (a–g), of which all but f contain actin as subunits. The mutant ida5 and a new strain, ida5-t, lack four subspecies (a, c, d, and e). These mutants were found to have mutations in the conventional actin gene, such that its product is totally lost; ida5 has a single-base deletion that results in a stop codon at a position about two-thirds from the 5′ end of the coding region, and ida5-t lacks a large portion of the entire actin gene. Two-dimensional gel electrophoresis patterns of the axonemes and inner-arm subspecies b and g of ida5 lacked the spot of actin (isoelectric point [pI] = ∼5.3) but had two novel spots with pIs of ∼5.6 and ∼5.7 instead. Western blot with different kinds of anti-actin antibodies suggested that the proteins responsible for the two novel spots and conventional actin are different but share some antigenicity. Since Chlamydomonas has been shown to have only a single copy of the conventional actin gene, it is likely that the novel spots in ida5 and ida5-t originated from another gene(s) that codes for a novel actin-like protein(s) (NAP), which has hitherto been undetected in wildtype cells. These mutants retain the two inner-arm subspecies b and g, in addition to f, possibly because NAP can functionally substitute for the actin in these subspecies while they cannot in other subspecies. The net growth rate of ida5 and ida5-t cells did not differ from that of wild type, but the mating efficiency was greatly reduced. This defect was apparently caused by deficient growth of the fertilization tubule. These results suggest that NAP can carry out some, but not all, functions performed by conventional actin in the cytoplasm and raise the possibility that Chlamydomonas can live without ordinary actin.
... The AChR a-subunit single-stranded probe (3) was synthesized from a DNA fragment of 500 nucleotides containing 120 nucleotides of exon P2 of the chicken AChR a-subunit gene (17) and labeled by a[32P]dATP (800 Ci/mmol; Amersham Corp.). The actin probe (giR of Dr. S. Alonso) was a 1,150-bp mouse eDNA insert in plasmid pAIA1 derived from cytoskeletal I~-aetin mRNA, as determined from its 3' untranslated sequence (1,24). It was labeled according to the Amersham multiprime procedure by a[32P]dCrP (Amersham Corp.; 800 Ci/mmol). ...
In cultured chicken myotubes, calcitonin gene-related peptide (CGRP), a peptide present in spinal cord motoneurons, increased by 1.5-fold the number of surface acetylcholine receptors (AChRs) and by threefold AChR alpha-subunit mRNA level without affecting the level of muscular alpha-actin mRNA. Cholera toxin (CT), an activator of adenylate cyclase, produced a similar effect, which did not add up with that of CGRP. In contrast, tetrodotoxin, a blocker of voltage-sensitive Na+ channels, elevated the level of AChR alpha-subunit mRNA on top of the increase caused by either CGRP or CT. 12-O-Tetradecanoyl phorbol-13-acetate (TPA), an activator of protein kinase C, markedly decreased the cell surface and total content of [125I]alpha BGT-binding sites and reduced the rate of appearance of AChR at the surface of the myotubes without reducing the level of AChR alpha-subunit mRNA. Moreover, TPA inhibited the increase of AChR alpha-subunit mRNA caused by tetrodotoxin without affecting that produced by CGRP or CT. Under the same conditions, TPA decreased the level of muscular alpha-actin mRNA and increased that of nonmuscular beta- and gamma-actins mRNA. These data suggest that distinct second messengers are involved in the regulation of AChR biosynthesis by CGRP and muscle activity and that these two pathways may contribute to the development of different patterns of AChR gene expression in junctional and extrajunctional areas of the muscle fiber.
... RNA was eleetrophoretically separated, blotted onto Zeta-Probe membrane (Bio-Rad Laboratories), UV cross-linked and probed in Church buffer (Church and Gilbert, 1984) at 65"C. The following oligolabeled eDNA probes were used: (a) p-5'6 corresponding to the 5' region of mouse tenasein; (b) M20/I corresponding to the middle region of mouse tenascin (Weller et al., 1991); (c) the Sph-Hind/lI fragment of eDNA clone for mouse laminin BI chain (Oberbiumer, 1986); (d) a clone for mouse ~lintegrin subunit detecting two polyadenylation variants of the mRNA; (e) a clone for mouse #-actin (Minty et al., 1983); (f) and a clone for human G3PDH (Clonteeh, Palo Alto, CA). Upon hybridization the blot with 3T3 RNA was washed twice for 30 rain at 650C in a solution containing 0.1 × SSC and 1% SDS. ...
Tenascin, a predominantly mesenchymal extracellular matrix (ECM) glycoprotein has a rather restricted tissue distribution, but until now factors that inhibit its expression have not been identified. Glucocorticoids are known to be beneficial for establishment of myelopoiesis in long-term bone marrow cultures. Tenascin was found to be expressed in the bone marrow, and glucocorticoids were found to affect bone marrow tenascin expression. Both tenascin mRNAs and the mRNA of another ECM protein, laminin B1 chain, were drastically downregulated by glucocorticoids during initiation of bone marrow cultures. However, in already established long-term cultures glucocorticoids did not affect laminin B1 chain mRNA levels although tenascin mRNAs continued to be downregulated. Studies with a stromal cell line (MC3T3-G2/PA6) and fibroblasts (3T3) suggested that glucocorticoids act directly on the stromal cells that produce tenascin. In 3T3 cells this downregulation occurred within 12 h of glucocorticoid-treatment, suggesting that glucocorticoids acted through cis regulatory elements of the tenascin gene. We suggest that glucocorticoids in part regulate hematopoiesis by modifying the ECM. Furthermore, downregulation of tenascin expression by glucocorticoids may in part explain the restricted tissue distribution of tenascin in other tissues.
... After hybridization, the membranes were processed by standard techniques and exposed to Kodak BioMax film (Eastman Kodak Co.) at Ϫ70°C with an intensifying screen. Labeled inserts used for Northern hybridizations were obtained as follows: the 0.38-kb BamHI/HindIII fragment of pGEMglo containing exon 2 of the rabbit -globin (Vakalopoulou et al., 1991), the 0.6-kb EcoRI fragment of pUK0321 containing part of the human u-PA cDNA (Cajot et al., 1989), the 1.4-kilobase pair EcoRI fragment of plasmid pJ7 containing the full-length cDNA encoding PAI-2 (Schleuning et al., 1987), the PstI fragment of plasmid pGEMPAI-1 encoding part of the human PAI-1 cDNA (Ny et al., 1986), the 2.5-kilobase pair BglII fragment of plasmid ptPA114B containing the full-length cDNA encoding human t-PA (Fisher et al., 1985), and the 1.4-kilobase pair PstI fragment of mouse -actin (Minty et al., 1983). ...
Plasminogen activator inhibitor type 2 (PAI-2) mRNA and antigen levels are synergistically induced in HT-1080 fibrosarcoma cells when treated with a combination of tumor necrosis factor (TNF) and phorbol 12-myristate 13-acetate (PMA). Here we demonstrate that this effect is not fully reflected at the level of gene transcription, suggesting a contribution of post-transcriptional events in this induction. Insertion of the 3′-untranslated region (3′-UTR) of PAI-2 mRNA into the 3′-UTR of a rabbit β-globin reporter gene reduces β-globin-PAI-2 chimeric mRNA expression in stably transfected cells. The region within the PAI-2 3′-UTR responsible for this effect is located within the 368-nucleotide sequence preceding the poly(A) tail, a segment that includes a nonameric UUAUUUAUU motif. Mutagenesis of this element abolishes the PAI-2 3′-UTR destabilizing effect, revealing a functional role for this motif. TNF and PMA co-treatment of transfected cells increases β-globin-PAI-2 chimeric mRNA expression 3-4-fold, indicating that the inherently unstable 3′-UTR of PAI-2 mRNA can become stabilized in response to TNF and PMA. Our results indicate that induction of PAI-2 gene expression by TNF and PMA involves both direct transcription as well as mRNA stabilization, the latter involving an AU-rich nonameric motif in the 3′-UTR.
... More than 20 actin-related sequences have been identified in vertebrate genomes by Southern blot analysis. Most of these sequences represent pseudogenes (Engel et al., 1982;Minty et al., 1983;Gunning et al. 1984;Warne et al., 1991). Evidence for as many as eight functional actins has been presented with four muscle and four nonmuscle isoforms Weber, 1978a,b, 1979a,b). ...
Smooth muscle γ-actin (SMGA) is an excellent marker of smooth muscle differentiation because it is essentially restricted to smooth muscle. As a first step toward unraveling the mechanisms underlying smooth muscle development and differentiation, we have examined the tissue-specific and developmental expression patterns of six constructs carrying portions of the murine SMGA gene linked to chloramphenicol acetyltransferase (CAT) in stable lines of transgenic mice. Based on the transgenic studies most, if not all, of the regulatory elements necessary for proper spatial and temporal expression of SMGA are present within a 13.7 kb segment of the SMGA gene containing 4.9 kb of upstream sequence, exon 1, intron 1, and a portion of exon 2 up to the start codon for translation. A second construct (SMGA11.6CAT) that lacks the distal 2.1 kb of upstream sequence but is otherwise identical to SMGA13.7CAT shows a similar level of smooth muscle-specific CAT activity. However, SMGA9.3CAT fusion gene containing only 571 bp of 5′ flanking sequence, but otherwise identical to SMGA13.7CAT, and SMGA6.0CAT containing only the 4.9 kb upstream sequence, exon 1, and a miniintron 1 show a more than a 100-fold reduction of CAT activity in most smooth muscle-rich tissues. Furthermore, removal of most or all of intron 1 from a transgene with 571 bp of upstream sequence (SMGA2.0 CAT and SMGA0.6CAT) results in a near-complete or complete loss of activity, respectively, in all tissues. Overall, the studies suggest that upstream elements between -2.7 kb and -571 bp and elements within intron 1 are required for high levels of SMGA gene expression in an appropriate temporal-spatial fashion. © 1996 Wiley-Liss, Inc.
... The probes used were the 1,000-bp Pst 1 insert of human PAIR cDNA (17) (kindly provided by Dr. David Loskutoff, Scripps Clinic and Research Foundation, La Jolla, CA) ; the Eco RI insert of pPAI J7, harboring 1,900 by of the human PAI-2 cDNA (18) ; the Bgl II fragment of pPA 11 4B cDNA harboring 1,948 by of the human t-PA cDNA (19) ; the Eco Rl fragment of pUK 0321 harboring 1,023 by of the human u-PA cDNA (15) . Mouse 0-actin cDNA (20) was a generous gift from Dr. Stefania Petrucco, Swiss Institute for Cancer Research, Epalinges, Switzerland ; a human genomic PAI-2 clone (chPAI-2 8) was isolated from a human Charon 4A gene library using the labeled Eco RI insert of pPAI J7 (above) . The authenticity of the clone was established by sequencing the first two exons . ...
... Isolation of actin cDNA clones. A Xgt-10 cDNA library, prepared from polyadenylated RNA isolated from purified residual bodies from CD-1 mice, was screened by plaque hybridization with a mouse 1-actin coding-region probe (pAL41) (34). Eight positive clones were isolated and characterized by restriction mapping. ...
A heavy form of DNA ligase (8S) replaces the light form (6S) of enzyme when Axolotl egg is fertilized or artificially activated at 18 °C. When eggs either fertilized or activated are maintained at 10 °C the heavy form of enzyme does not appear and the light DNA ligase remains undiminished.
Genes representing six different actin isoforms were isolated from a chicken genomic library. Cloned actin cDNAs as well as tissue-specific mRNAs enriched in different actin species were used as hybridization probes to group individual actin genomic clones by their relative thermal stability. Restriction maps showed that these actin genes were derived from separate and nonoverlapping regions of genomic DNA. Of the six isolated genes, five included sequences from both the 5' and 3' ends of the actin-coding area. Amino acid sequence analysis from both the NH2- and COOH-terminal regions provided for the unequivocal identification of these genes. The striated isoforms were represented by the isolated alpha-skeletal, alpha-cardiac, and alpha-smooth muscle actin genes. The nonmuscle isoforms included the beta-cytoplasmic actin gene and an actin gene fragment which lacked the 5' coding and flanking sequence; presumably, this region of DNA was removed from this gene during construction of the genomic library. Unexpectedly, a third nonmuscle chicken actin gene was found which resembled the amphibian type 5 actin isoform (J. Vandekerckhove, W. W. Franke, and K. Weber, J. Mol. Biol., 152:413-426). This nonmuscle actin type has not been previously detected in warm-blooded vertebrates. We showed that interspersed, repeated DNA sequences closely flanked the alpha-skeletal, alpha-cardiac, beta-, and type 5-like actin genes. The repeated DNA sequences which surround the alpha-skeletal actin-coding regions were not related to repetitious DNA located on the other actin genes. Analysis of genomic DNA blots showed that the chicken actin multigene family was represented by 8 to 10 separate coding loci. The six isolated actin genes corresponded to 7 of 11 genomic EcoRI fragments. Only the alpha-smooth muscle actin gene was shown to be split by an EcoRI site. Thus, in the chicken genome each actin isoform appeared to be encoded by a single gene.
Using several actin isotype-specific cDNA probes, we found actin mRNA of two size classes, 2.1 and 1.5 kilobases (kb), in extracts of polyadenylated and nonpolyadenylated RNA from sexually mature CD-1 mouse testes. Although the 2.1-kb sequence was present in both meiotic and postmeiotic testicular cell types, it decreased manyfold in late haploid cells. The 1.5-kb actin sequence was not detectable in meiotic pachytene spermatocytes (or in liver or kidney cells), but was present in round and elongating spermatids and residual bodies. To differentiate between the beta- and gamma-actin mRNAs, we isolated a cDNA, pMGA, containing the 3' untranslated region of a mouse cytoplasmic actin that has homology to the 3' untranslated region of a human gamma-actin cDNA but not to the 3' untranslated regions of human alpha-, beta-, or cardiac actins. Dot blot hybridizations with pMGA detected high levels of presumptive gamma-actin mRNA in pachytene spermatocytes and round spermatids, with lower amounts found in elongating spermatids. Hybridization with the 3' untranslated region of a rat beta-actin probe revealed that round spermatids contained higher levels of beta-actin mRNA than did pachytene spermatocytes or residual bodies. Both probes hybridized to the 2.1-kb actin mRNA but failed to hybridize to the 1.5-kb mRNA.
We identified a novel chicken actin gene. The actin protein deduced from its nucleotide sequence very closely resembles the vertebrate cytoplasmic actins; accordingly, we classified this gene as a nonmuscle type. We adopted the convention for indicating the nonmuscle actins of the class Amphibia (Vandekerckhove et al., J. Mol. Biol. 152:413-426) and denoted this gene as type 5. RNA blot analysis demonstrated that the type 5 actin mRNA transcripts accumulate in adult tissues in a pattern indicative of a nonmuscle actin gene. Genomic DNA blots indicated that the type 5 actin is a single copy gene and a distinct member of the chicken actin multigene family. Inspection of the nucleotide sequence revealed many features that distinguished the type 5 gene from all other vertebrate actin genes examined to date. These unique characteristics include: (i) an initiation Met codon preceding an Ala codon, a feature previously known only in plant actins, (ii) a single intron within the 5' untranslated region, with no interruptions in the coding portion of the gene, and (iii) an atypical Goldberg-Hogness box (ATAGAA) preceding the mRNA initiation terminus. These unusual features have interesting implications for actin gene diversification during evolution.
Transfection into cultured cell lines was used to investigate the transcriptional regulation of the human cardiac actin gene. We first demonstrated that in both human heart and human skeletal muscle, cardiac actin mRNAs initiate at the identical site and contain the same first exon, which is separated from the first coding exon by an intron of 700 base pairs. A region of 485 base pairs upstream from the transcription initiation site of the human cardiac actin gene directs high-level transient expression of the bacterial chloramphenicol acetyltransferase gene in differentiated myotubes of the mouse C2C12 muscle cell line, but not in mouse L fibroblast or rat PC-G2 pheochromocytoma cells. Deletion analysis of this region showed that at least two physically separated sequence elements are involved, a distal one starting between -443 and -395 and a proximal one starting between -177 and -118, and suggested that these sequences interact with positively acting transcriptional factors in muscle cells. When these two sequence elements are inserted separately upstream of a heterologous (simian virus 40) promoter, they do not affect transcription but do give a small (four- to fivefold) stimulation when tested together. Overall, these regulatory regions upstream of the cap site of the human cardiac actin gene show remarkably high sequence conservation with the equivalent regions of the mouse and chick genes. Furthermore, there is an evolutionarily conserved repeated motif that may be important in the transcriptional regulation of actin and other contractile protein genes.
The complete nucleotide sequence of a genomic clone encoding the mouse skeletal alpha-actin gene has been determined. This single-copy gene codes for a protein identical in primary sequence to the rabbit skeletal alpha-actin. It has a large intron in the 5'-untranslated region 12 nucleotides upstream from the initiator ATG and five small introns in the coding region at codons specifying amino acids 41/42, 150, 204, 267, and 327/328. These intron positions are identical to those for the corresponding genes of chickens and rats. Similar to other skeletal alpha-actin genes, the nucleotide sequence codes for two amino acids, Met-Cys, preceding the known N-terminal Asp of the mature protein. Comparison of the nucleotide sequences of rat, mouse, chicken, and human skeletal muscle alpha-actin genes reveals conserved sequences (some not previously noted) outside of the protein-coding region. Furthermore, several inverted repeat sequences, partially within these conserved regions, have been identified. These sequences are not present in the vertebrate cytoskeletal beta-actin genes. The strong conservation of the inverted repeat sequences suggests that they may have a role in the tissue-specific expression of skeletal alpha-actin genes.
In the mouse mammary tumor virus promoter, a tandem of octamer motifs, recognized by ubiquitous and tissue-restricted Oct transcription factors, is located upstream of the TATA box and next to a binding site for the transcription factor nuclear factor I (NF-I). Their function was investigated with mutant long terminal repeats under different transfection conditions in mouse Ltk- cells and quantitative S1 nuclease mapping of the transcripts. In stable transfectants, which are most representative of the state of proviral DNA with respect to both number of integrated DNA templates and chromatin organization, a long terminal repeat mutant of both octamer sites showed an average 50-fold reduction of the basal transcription level, while the dexamethasone-stimulated level was unaffected. DNase I in vitro footprinting assays with L-cell nuclear protein extracts showed that the mutant DNA was unable to bind octamer factors but had a normal footprint in the NF-I site. I conclude that mouse mammary tumor virus employs the tandem octamer motifs of the viral promoter, recognized by the ubiquitous transcription factor Oct-1, for its basal transcriptional activity and the NF-I binding site, as previously shown, for glucocorticoid-stimulated transcription. A deletion mutant with only one octamer site showed a marked base-level reduction at high copy number but little reduction at low copies of integrated plasmids. The observed transcription levels may depend both on the relative ratio of transcription factors to DNA templates and on the relative affinity of binding sites, as determined by oligonucleotide competition footprinting.
We have studied the expression of the alpha-amylase, trypsin, and elastase II genes in the acinar pancreas during mouse development. Transcriptional control is the major mechanism by which the differential accumulation of alpha-amylase, trypsin, and elastase II mRNAs is determined during late embryogenesis. The synthesis of pancreatic mRNAs is detected around day 15 of gestation and involves most if not all acinar cells. The DNA-binding activity of the pancreas-specific transcription factor PTF1, which binds to enhancers of genes expressed in this tissue, is detected for the first time at day 15 of gestation. The appearance of the factor at this early stage of development suggests that it plays an important role during pancreas differentiation.
We have produced seven lines of transgenic mice carrying the quail gene encoding the fast skeletal muscle-specific isoform of troponin I (TnIf). The quail DNA included the entire TnIf gene, 530 base pairs of 5'-flanking DNA, and 1.5 kilobase pairs of 3'-flanking DNA. In all seven transgenic lines, normally initiated and processed quail TnIf mRNA was expressed in skeletal muscle, where it accumulated to levels comparable to that in quail muscle. Moreover, in the three lines tested, quail TnIf mRNA levels were manyfold higher in a fast skeletal muscle (gastrocnemius) than in a slow skeletal muscle (soleus). We conclude that the cellular mechanisms directing muscle fiber type-specific TnIf gene expression are mediated by cis-regulatory elements present on the introduced quail DNA fragment and that they control TnIf expression by affecting the accumulation of TnIf mRNA. These elements have been functionally conserved since the evolutionary divergence of birds and mammals, despite the major physiological and morphological differences existing between avian (tonic) and mammalian (twitch) slow muscles. In lines of transgenic mice carrying multiple tandemly repeated copies of the transgene, an aberrant quail TnIf transcript (differing from normal TnIf mRNA upstream of exon 2) also accumulated in certain tissues, particularly lung, brain, spleen, and heart tissues. However, this aberrant transcript was not detected in a transgenic line which carries only a single copy of the quail gene.
In Trypanosoma brucei, the actin gene is present in a cluster of two, three, or four tandemly linked copies, depending on the strain. Each cluster seems to exist in two allelic versions, as suggested by the polymorphism of both gene number and restriction fragment length in the DNA from cloned trypanosomes. The amplification of the gene copy number probably occurs through unequal sister chromatid exchange. The chromosomes harboring the actin genes belong to the large size class. The coding sequence was 1,128 nucleotides long and showed 60 to 70% homology to other eucaryotic actin genes. Surprisingly, this homology seemed weaker with Trypanosoma congolense, Trypanosoma cruzi, Trypanosoma vivax, Trypanosoma mega, or Leishmania actin-specific sequences. The mRNA was around 1.6 kilobases long and was synthesized at the same level in bloodstream and procyclic forms of the parasite. Large RNA precursors, up to 7.7 kilobases, were found in a pattern identical in strains containing either two or three gene copies. Probing of the flanking regions of the gene with either steady-state or in vitro transcripts, as well as S1 nuclease protection and primer extension experiments, allowed mapping of the 3' splice site of the actin mRNA, 38 nucleotides upstream from the translation initiation codon. A variably sized poly(dT) tract was found about 30 base pairs ahead of the splice site. The largest detected actin mRNA precursor seemed to give rise to at least two additional stable mRNAs. The RNA polymerase transcribing the actin gene exhibited the same sensitivity to inhibition by alpha-amanitin as that transcribing both the spliced leader and the bulk of polyadenylated mRNAs.
We described the structures of mouse cytoskeletal gamma-actin cDNA clones and showed that there is strong conservation of the untranslated regions with human gamma-actin cDNA. In addition, we found that the expression levels of beta- and gamma-actin mRNAs are differentially controlled in various mouse tissues and cell types but are coordinately increased in the cellular growing state. These results suggest that there are multiple regulatory mechanisms of cytoskeletal actin genes and are consistent with the argument that beta- and gamma-actins might have functional diversity in mammalian cells.
Hybridization to synthetic oligonucleotides representing conserved regions in the promoter and first intron of several vertebrate beta-actin genes was used to discriminate between what appears to be a single functional beta-actin gene and numerous pseudogenes in the mouse genome. Sequences derived from the 5' end of this gene were shown to confer serum-inducible expression upon a heterologous reporter gene when transfected into mouse fibroblasts. Moreover, these sequences rendered reporter gene expression superinducible by a combination of serum and cycloheximide. These experiments indicate that the 5' end of the mouse beta-actin gene contains sequence elements which mediate the stimulatory effects of serum growth factors and which are responsive to both positive and negative regulators of gene expression.
We have constructed isotype-specific subclones from the 3' untranslated regions of alpha-skeletal, alpha-cardiac, beta-cytoskeletal, and gamma-cytoskeletal actin cDNAs. These clones have been used as hybridization probes to assay the number and organization of these actin isotypes in the human genome. Hybridization of these probes to human genomic actin clones (Engel et al., Proc. Natl. Acad. Sci. U.S.A. 78:4674-4678, 1981; Engel et al., Mol. Cell. Biol. 2:674-684, 1982) has allowed the unambiguous assignment of the genomic clones to isotypically defined actin subfamilies. In addition, only one isotype-specific probe hybridizes to each actin-containing gene, with a single exception. This result suggests that the multiple actin genes in the human genome are not closely linked. Genomic DNA blots probed with these subclones under stringent conditions demonstrate that the alpha-skeletal and alpha-cardiac muscle actin genes are single copy, whereas the cytoskeletal actins, beta and gamma, are present in multiple copies in the human genome. Most of the actin genes of other mammals are cytoplasmic as well. These observations have important implications for the evolution of multigene families.
Mouse testis contains two size classes of actin mRNAs of 2.1 and 1.5 kilobases (kb). The 2.1-kb actin mRNA codes for cytoplasmic beta- and gamma-actin and is found throughout spermatogenesis, while the 1.5-kb actin mRNA is first detected in postmeiotic cells. Here we identify the testicular postmeiotic actin encoded by the 1.5-kb mRNA as a smooth-muscle gamma-actin (SMGA) and present its cDNA sequence. The amino acid sequence deduced from the postmeiotic actin cDNA sequence was nearly identical to that of a chicken gizzard SMGA, with one amino acid replacement at amino acid 359, where glutamine was substituted for proline. The nucleotide sequence of the untranslated region of the SMGA differed substantially from those of other isotypes of mammalian actins. By using the 3' untranslated region of the testicular SMGA, a highly specific probe was obtained. The 1.5-kb mRNA was detected in RNA from mouse aorta, small intestine, and uterus, but not in RNA isolated from mouse brain, heart, and spleen. Testicular SMGA mRNA was first detected and increased substantially in amount during spermiogenesis in the germ cells, in contrast to the decrease of the cytoplasmic beta- and gamma-actin mRNAs towards the end of spermatogenesis. Testicular SMGA mRNA was present in the polysome fractions, indicating that it was translated. These studies demonstrate the existence of an SMGA in male haploid germ cells. The implications of the existence of an SMGA in male germ cells are discussed.
A cDNA library was constructed from HL60 human promyelocyte poly(A)+ RNA harvested 3 h after induction of macrophage differentiation with 12-O-tetradecanoyl phorbol-13-acetate in the presence of cycloheximide. We isolated from this library a 1.6-kilobase full-length clone designated b4 whose corresponding mRNA was greatly increased in abundance in cytoplasmic RNA under these conditions. Dideoxy sequencing revealed that this mRNA encoded MONAP (monocyte-derived neutrophil-activating peptide), a 10-kilodalton monokine with neutrophil-specific chemotactic and enzyme-releasing activities. The 3' untranslated region of this mRNA was found to be 1.2 kilobases long and possessed nine copies of the AUUUA sequence known to be associated with regulation of mRNA stability. Actinomycin D chase experiments yielded evidence that cytoplasmic stabilization was one of the means of regulation of MONAP expression. Analysis of cytoplasmic poly(A)- RNA revealed the presence of several discrete truncated species that shared a common 5' end and appeared to be intermediates of degradation. S1 mapping showed that the 3' ends of these molecules were distributed throughout the 3' untranslated region, preferentially in A + U-rich regions, broadly correlating with the distribution of AUUUA sites. Nuclear run-on experiments indicated that transcriptional induction accounted for less than 15% of the accumulation of MONAP mRNA. This mRNA was induced in HL60 cells by treatment with several differentiation-inducing agents: 12-O-tetradecanoyl phorbol-13-myristate alone, sodium butyrate, vitamin D3, and dimethyl sulfoxide. It was also induced in quiescent diploid lung fibroblasts stimulated to divide by serum, and it was constitutively overexpressed by some human tumor lines.
Transcription of the mouse mammary tumor virus DNA is known to be induced by several steroid hormones. Using chimeric MMTV plasmids containing mutations within the hormone regulatory element, we have previously studied the regions required for the glucocorticoid response in mouse fibroblasts. Here we report the characterization of elements essential for the stimulation by progestins and androgens as compared with glucocorticoids. The same set of mutant plasmids was transfected into the human mammary tumor cell line T47D, and the specific transcripts were analyzed by an S1 nuclease protection assay. Androgen-mediated stimulation, although weak, showed an extended sensitivity to mutations, with a slight preference for the proximal region. The results with progestin suggest that sequences within all the described sites protected by the receptor in vitro are required and that the promoter-proximal region (-128 to -78 from the RNA start site) is more important than the distal one (-190 to -160). Moreover, a binding site for nuclear factor I was not required for the progestin response, whereas it was required for glucocorticoids. Thus, the various steroid receptors play a role in the differential regulation of mouse mammary tumor virus transcription by recognizing distinct sequence differences in the hormone regulatory element and interacting with different factors bound to the promoter.
Now that the sequence of the mouse genome is completely known, the position of any gene of the species can be accurately and rapidly established by searching the appropriate database. In this context, a chapter devoted to gene mapping and genetic maps might appear somewhat outdated, not to say useless. However, we thought that it might be interesting to reconsider this subject for at least three reasons. The first is that gene mapping has been a major component of the activities of mouse geneticists during most of the twentieth century; it is then interesting, if only from a historical point of view, to briefly describe the techniques and methods that have made the genetic map of the mouse the richest and most documented map of all mammals, including humans, for nearly 50 years. The second reason is more fundamental and refers to the many mutations that occur spontaneously in the breeding nuclei of inbred strains or those that are induced by mutagenic agents. All these mutations are initially characterized by an abnormal phenotype and some of them may appear of potential interest, for example, as models of human diseases. However, annotating and characterizing all these mutations requires that they be first carefully located on a chromosome and analyzed at the molecular level, when relevant.
The process of myelination involves the coordinated expression of specific genes in oligodendrocytes and Schwann cells. Since the formation of myelin is the result of an interaction of one of the above cell types with an appropriate class of axon, it might be predicted that regulatory signals exist which allow for the recognition of the correct axonal type and specify the induction of expression of myelin-specific genes. A number of lines of evidence infer the existence of such signals, but the biochemical identity of the signals, and the mechanisms and pathways through which they act, remain unknown. In this article I shall explore what we can infer about this hypothesized regulation, and present some experimental approaches which may shed light on the questions raised.
Mechanical overload is usually tolerated over a long period because of the development of several adaptational factors. At the fiber level, the reaction of myocardium to chronic overload is basically the same as that of skeletal muscle [1]. Two fundamental processes help striated muscle to adapt to a chronic enhancement of work: a decrease in the maximum shortening velocity for an unloaded muscle which in turn improves efficiency, and a hypertrophy which adapts the tissue to the new requirements both by multiplying the contractile units and, for the heart, by lowering the wall stress [2].
Objectives: To define the chronology of protein synthesis during hepatic regeneration and to verify the possibilities of stimulation. Methods: 70% hepatectomy was performed in 3 groups of 33 rats: a control group, one group receiving 15 μg/kg/day of Epidermal Growth Factor (day-1 to day 14) and one group receiving 4 μg/day of interleukin 6 (day 1 to day 14). m RNAs coding for protooncogene c-Myc, α-foetoprotein, α2-macroglobulin, actin, albumin, heat shock protein, glucose-6-phosphate dehydrogenase, were quantified after sacrifice scheduled from the 30(th) minute to the 14(th) day following hepatectomy. Results were interpreted after analysis of variance. Results: 1) Controls: mRNA concentration of α2-macroglobulin and α-foetoprotein increased, by 20 and 10 times the baseline level respectively at the 20(th) hour and c-Myc mRNA increased by 10 times at the 72(nd) hour. The mRNA concentration of other proteins did not change during the experiments. 2) Epidermal Growth Factor had no stimulating effect when compared with controls. 3) The mRNA concentration of α2-macroglobulin reached 40 times the baseline level at the 20(th) hour when interleukin 6 was used while that of heat shock protein significantly increased at the 72(nd) hour. These variations were different from those of the controls. The mRNAs of other proteins did not vary. Conclusion: Reconstructive processes are activated early; the remnant liver is receptive to stimulation; Epidermal Growth Factor had no effect; interleukin 6 stimulates reconstructive processes.
The word cytoskeleton is the most commonly used term indicating microtubules (MT), microfilaments and 10-nm filaments in a structural and functional system. Because we have learned that these self-assembly structures also work as elements of intracellular transport and cell movement, this term has become rather inaccurate. Considering these functions, DE DUVE (1984) used the terms cytobones and cytomuscles, both of which much better characterize the tasks which MT and microfilaments are known to fulfill in animal, fungal, and plant cells. However, the functional diversity of these structures makes it impossible to designate them by the use of one or two words only. Therefore, cytoskeleton will be further used in this article, but only as a working expression.
In this review, evolutionary relationships of actin gene and protein sequences, as well as intron positions within actin genes, are analyzed both within and between eukaryotic kingdoms.
We asked whether nonlethal injury to the oligodendrocyte as manifested by altered myelin gene expression is an early event in the pathogenesis of demyelinating disease and subsequent remyelination. Using simultaneous in situ hybridization and immunocytochemistry, we studied expression of proteolipid protein (PLP) antigen and mRNA in spinal cords of normal adult mice and of mice infected with Theiler's virus which provides an excellent model for multiple sclerosis. Downregulation of PLP mRNA was observed within 3 days and persisted for as long as 367 days following intracerebral virus infection of SJL/J mice which are susceptible to chronic demyelination. Downregulation of myelin gene products preceded the development of prominent inflammation and demyelination observed following virus infection. In contrast, no change from control uninfected mice was observed in the expression of PLP mRNA following infection of C57BL/10SNJ mice which are resistant to demyelination. Treatment of chronically infected susceptible SJL/J mice with a regimen which promotes CNS-type (oligodendroglial) remyelination resulted in a 3- to 4-fold increase in PLP mRNA expression in oligodendrocytes. Actin mRNA expression in PLP antigen-positive cells was unchanged following TMEV-induced demyelination or remyelination indicating up- or downregulation of myelin gene products as compared to constitutively expressed actin gene. These experiments support the hypothesis that early regulation of myelin gene expression may be an important determinant in demyelination and in remyelination following nonlethal injury to oligodendrocytes.
A β-actin gene of carp (Cyprinus carpio) was isolated from a genomic EMBL3 library. The nucleotide sequence of the gene indicates six exons spanning 3.6 kb. Southern blot hybridization of restriction endonuclease digests of carp genomic DNA indicate that there are two copies of the β-actin isotype and several other species of actin genes. The transcriptional start site is 85 bp and 24 bp downstream respectively from consensus CCAAT and TATA promoter elements. The organization of the carp β-actin gene is identical to that of chicken, human, and rat genes in terms of size, exon/intron locations and junctions and in having a translationally silent first exon. The fish gene is 90% and 99% conserved at the nucleotide and amino acid levels, respectively, with land vertebrate β-actin genes. Northern blot analysis of β-actin gene expression indicated that the gene is highly expressed in brain, less so in muscle, and much less so in liver cells. The putative β-actin proximal promoter of carp, identified by the con...
A human actin cDNA clone pGF3 isolated from a fetal skeletal muscle cDNA library is described. The insert cDNA is homologous to skeletal muscle a-actin as judged by restriction mapping and nucleotide sequencing. The recombinant contains a substantial portion of the coding and the complete 3'-untranslated region. Comparison of the 3' ends of human and rat skeletal muscle and human cardiac a-actins reveals little homology between different types of actin genes in man but marked conservation of this region in the skeletal muscle actins of man and rat.
We have explored the effect of insulin on the modulation of mRNA and antigen levels of components of the fibrinolytic enzyme system in human hepatoma (Hep G2) and fibrosarcoma (HT-1080) cells. Treatment of Hep G2 cells with a physiological concentration of insulin provokes a 2-fold increase in plasminogen activator inhibitor (PAI)-1 antigen. A similar increase is observed for both the 3.2 and 2.3 kb transcripts of PAI-1 mRNA. Cycloheximide when added alone to cells preferentially suppresses constitutive expression of the 3.2 kb PAI-1 mRNA and blocks insulin-mediated induction of both 3.2 and 2.3kb PAI-1 mRNA. There was no detectable expression of urokinase (u-PA) or tissue-type plasminogen activator (t-PA) antigen or mRNA in these cells under constitutive conditions or after treatment with insulin. In HT-1080 cells, basal expression of PAI-1, t-PA and u-PA mRNA and antigen was not modulated by insulin treatment. The results from these experiments indicate firstly, that induction of PAI-1 by insulin requires on-going protein biosynthesis, and secondly, that the two species of PAI-1 mRNA have a different requirement for protein biosynthesis.
Background
TrkA mRNA expression has been reported to be related to favorable outcome of neuroblastoma (NB). Previously, we found that interferon-γ (IFN-γ) can enhance TrkA mRNA expression in NB cell lines. In the present study, we examined the effect of nerve growth factor (NGF) on IFN-γ-induced TrkA protein to clarify the relationship between TrkA and cell differentiation of NB.ProcedureThe effect of IFN-γ on the TrkA mRNA expression was screened in six human NB cell lines and a freshly prepared sample, SK-rib, from a stage IV patient. Using two of them, we examined their morphological change during simultaneous loading of NGF and IFN-γ. Tyrosine phosphorylation pattern after 5 min of NGF stimulation was also examined in immunoblot analysis with anti-gp140trkA antibody and antiphospho tyrosine antibody.ResultsAfter a 4-day treatment with 500 IU/ml IFN-γ, TrkA mRNA increased in five cell lines and SK-rib cells in association with growth inhibition. Although the degree of morphological differentiation did not increase in proportion to the TrkA expression induced by IFN-γ, continuous loading of both IFN-γ and NGF caused marked morphological differentiation in a cultured KP-N-RT cell line and SK-rib cells during 10 days. Moreover, 5 min of NGF stimulation after IFN-γ treatment caused the phosphorylation of TrkA protein and downstream proteins.ConclusionsIFN-γ could induce the functional NGF receptor even in the aggressive phenotype of NB. Med. Pediatr. Oncol. 34:394–401, 2000. © 2000 Wiley-Liss, Inc.
Tumor necrosis factor alpha (TNF alpha) is likely to exert a major influence in the pathogenesis of glomerulopathies. Besides its proinflammatory properties. TNF alpha interacts with cell growth and synthesis of components of the fibrinolytic system. In this study, we report the effects of recombinant human TNF alpha on the synthesis of tissue-type plasminogen activator (t-PA) and its inhibitor (PAI-1) by human mesangial cells in culture. We first demonstrate that TNF alpha binds specifically to a single class of high affinity receptors (Kd 5.10(-11) M; 1500 receptors/cell). TNF alpha has an antimitogenic effect on human mesangial cells since it decreased DNA synthesis, measured by 3H-thymidine incorporation, in a dose-dependent manner. Release of cytosolic LDH and incorporated 51Cr was not increased by 100 ng/ml TNF alpha as compared with control, indicating that this monokine is not cytotoxic for cultured human mesangial cells. Zymographic analysis and reverse fibrin autography disclosed a 120 kD t-PA-PAI-1 complex and a 50 kD free form of PAI-1 in the supernatants of both unstimulated and TNF-stimulated cells; PAI-1 was released in excess and free t-PA was not observed. TNF alpha (0 to 100 ng/ml) had no effect on t-PA synthesis, but enhanced PAI-1 release in a time- and dose-dependent manner (97% increase of PAI-1 synthesis after a 24 hour incubation). This effect was abolished by cycloheximide, suggesting that protein synthesis was required. Northern blot analysis showed that TNF alpha increased the steady-state PAI-1 mRNA levels in a time-dependent manner, with a maximal effect at two hours.(ABSTRACT TRUNCATED AT 250 WORDS)
DMA from ten mouse genomic clones, each containing distinct γ -actin processed pseudogenes, was subjected to electron microscopic
heteroduplex analysis, and in three cases (λmA36, λmAll8 and λmAll9) the heteroduplex formed with the DNA of a reference clone
was found to be interrupted by a single-stranded loop. The genomic regions corresponding to these loops were subjected to
structural analysis and they were found to represent different elements (IEs) inserted into the pseudogenes in a manner that
gave rise to short target-site direct repeats. IE 36 (500 base-pairs in length) was found to be an intercisternal A-particle
solo long terminal repeat (LTR), a 46 nucleotide region of which had undergone five-fold tandem amplification and subsequent
mutation. IE 119 (501 base-pairs in length) was also a solo LTR, bearing similarity to the recently-described GLN-3 class
of murine retroviral-like elements. IE 118 (865 base-pairs in length) is repeated 1000–2000 times in the mouse genome. It
is not related to any known class of mobile elements, but does possess some sequence motifs that suggest it may be an LTR
of a hitherto unrecognised family of retroviral-like elements. It also possesses a 26 out of 27 nucleotide identity to a region
of the flanking pseudogene, suggesting that it may have suffered gene conversion.
We sequenced the entire chicken α-cardiac actin gene. A single intron was positioned 20 bp upstream from the initiation ATG
codon in the 5′ non-coding region while the coding region was interrupted by 5 introns at amino acid positions 41/42, 150,
204, 267, and 327/328.Sequencing allowed the first comparison of the α-cardiac and α-skeletal actin transcriptional promoters.
These highly G+C rich promoters share two regions of homology which are found at position −134 (10 bp) end −296 (12 bp) in
the α-cardiac actin promoter. A smaller 9 bp motif (CCGCCCCGG) homologous to the −134 sequence was detected before, between
and after the TATA and CAAT boxes of the α-cardiac actin gene. The polyadenylation signal (AATAAA) was located 156 bp downstream
from the translation termination codon. The complete length of the α-cardiac actin mRNA excluding the poly A tail is 1370
nucleotides. The 3′ noncoding transcribed portion of the chicken α-cardiac actin gene was found to be extraordinarily conserved
when compared to the human and rat α-cardiac actin mRNA sequences.
The synthesis of the cytoskeletal protein actin exhibits, in the rat hypothalamus, a diurnal variation with maxima during morning hours. The objective of the present study was to assess whether melatonin injection could affect the in vitro incorporation of 35S-methionine into actin, as well as the levels of actin mRNA, in the hypothalamus of adult male rats treated either acutely or chronically with the hormone at 10: 00 or 18: 00. Injection of 100 μg/kg of melatonin for ten days at either time induced a significant depression in the incorporation of 35S-methionine into a 43 kDa protein with the electrophoretic mobility of actin. The specific activity of total soluble proteins after labeled methionine incubations decreased only after evening melatonin administration (100 μg/kg, ten days). Hypothalamic actin mRNA levels, quantitated by dot-blot analysis, decreased only after the injection of 100 μg/kg melatonin for ten days at 10:00. Neither a 10-μg/kg dose of melatonin, nor a single injection of 100 μg/kg melatonin, caused any significant change in the parameters examined. Melatonin (100 μg/kg for ten days) did not modify hypothalamic somatostatin or H-Ras mRNA concentration. These results suggest the existence of an inhibitory effect of melatonin on hypothalamic actin synthesis.
Three human neuroblastoma cell lines were examined to determine the effect of recombinant γ-interferon (IFN-γ) treatment on the expression of trk proto-oncogene. Increased levels of trk proto-oncogene mRNA were observed in two neuroblastoma cell lines (KP-N-RT and KP-N-SI(FA)) after IFN-γ treatment. The levels of trk mRNA increased with growth inhibition and morphological change in a time- and dose-dependent manner. The decreased level of N-myc mRNA after IFN-γ-treatment in KP-N-RT was inversely correlated with trk mRNA. Our results suggest that IFN-γ can modulate the signal transduction of nerve growth factor in human neuroblastoma cells.
cDNA probes for human retinoic acid receptors α and β (RARα and RARβ) were modified for use as specific hybridization probes to study hepatocellular carcinomas (HCC) and cell lines, liver regeneration, and fetal development. RARβ mRNA was detected at low levels in adult liver and rose markedly during the early phase of liver regeneration. RARβ mRNA was present at very low levels in HCC and was not detected in fetal liver. In contrast, RARα mRNA was present at low levels in normal liver, but showed a marked elevation in several HCCs and cell lines. Growth of cell lines was altered by retinoic acid (RA), but the effects could not be predicted by the levels of either RARα or RARβ mRNA. However, the response correlated with cell phenotype. Three cell lines with an adult phenotype (high albumin and low α-fetoprotein) were inhibited by RA, two undifferentiated lines showed moderate growth stimulation, and two of three cell lines that had high levels of α-fetoprotein were markedly stimulated by RA.
The nudeotide sequence of the chick β-actin gene was determined. The gene contains 5 introns; 4 interrupt the translated region
at codons 41/42, 120/122, 267, 327/328 and a large intron occurs in the 5′ untranslated region. The gene has a 97 nudeotide
5 ′-untrtranslated region and a 594 nudeotide 3′-untranslated region. A slight heterogeneity in the position of the poly A
addition site exists; polyadenylation can occur at either of two positions two nucleotides apart. The gene codes for an mRNA
of 1814 or 1816 nucleotides, excluding the poly(A) tail. In contrast to the chicle skeletal muscle actin gene the β-actin
gene lacks the Cys codon between the initiator ATG and the codon for the N-terminal amino acid of the mature protein. la the
5′ flanking DNA, 15 nucleotides downstream from the CCAAT sequence, is a tract of 25 nucleotides that is highly homologous
to the sequence found in the same region of the rat β-actin gene.
Thecomplete nucleotide sequence ofagenomic clone encoding themouseskeletal a-actin genehasbeen determined. Thissingle-copy genecodes foraprotein identical inprimary sequence totherabbit skeletal a-actin. Ithasalarge intron inthe5'-untranslated region 12nucleotides upstream fromtheinitiator ATGand five small introns inthecoding region atcodons specifying aminoacids 41/42, 150, 204, 267, and327/328. These intron positions areidentical tothose forthecorresponding genes ofchickens andrats. Similar toother skeletal a-actin genes, thenucleotide sequence codes fortwoaminoacids, Met-Cys, preceding theknownN-terminal Aspofthemature protein. Comparison ofthenucleotide sequences ofrat, mouse, chicken, andhumanskeletal muscle a-actin genes reveals conserved sequences (some notpreviously noted) outside oftheprotein-coding region. Furthermore, several inverted repeat sequences, partially within these conserved regions, havebeen identified. These sequences arenotpresent inthevertebrate cytoskeletal (-actin genes. Thestrong conservation oftheinverted repeat sequences suggests that they mayhavearole inthetissue-specific expression ofskeletal a-actin genes. Theactins represent amultigene family ofhighly con- served proteins found inall eucaryotes. Differences inamino acid sequence amongthevarious actins haveshownthat at least sixdifferent isoforms areexpressed invertebrates (52, 53). Twostriated muscle isoforms, skeletal aandcardiac a (52), andtwosmoothmuscle isoforms (53) arefoundinthe contractile apparatus ofmuscle fibers, whereas twocyto- skeletal isoforms, pandy,arepresent inthecytoskeleton of all cells (51). Theseactin proteins areextremely conserved inaminoacid sequence. Actin geneexpression istissue specific anddevelopmen- tally regulated (27, 29,32,35). Bystudying thestructural organization oftheactin genefamily, onecanbegin tolook forthecontrolling elements whichmodulate theexpression ofthese genesduring development. Herewe present the complete nucleotide sequence ofthesingle genomic copyof themouseskeletal a-actin gene. Thecoding region ofthis geneisinterrupted byfive introns whicharelocated inthe samepositions asintrons previously identified inother vertebrate skeletal a-actin genes(13, 54). A comparison of thenucleotide sequences ofseveral vertebrate skeletal a- actin genesreveals several blocks ofhighly conserved sequences inthe5'-flanking region andinboththe5'-and 3'-untranslated regions. Interestingly, theconserved se- quencesinthe5'-flanking region andwithin thefirst untranslated exoncanpotentially formseveral hairpin loops bybasepairing between adjacent inverted complementary sequences. Theseregions donotcorrespond tothepotential hairpin structure inthecorresponding portion oftherat cytoskeletal ,B-actin gene(33). Furthermore, itispossible to formlonghairpin loops within thefirst intron andonestem loop inthe3'-untranslated region upstream fromtheputative polyadenylation signal ATTAAA.Theseinteresting second- arystructures areapparently notpresent inthevertebrate cytoskeletal P-actin genes. Toourknowledge, this isthefirst description ofpotential secondary structures inthefirst
Plasmid DNAs from six strains of Saccharomyces cerevisiae were compared. Three different plasmids were found, designated Scp 1, Scp 2 and Scp 3, with monomer lengths of 6. 19, 6.06
and 5.97 kilobases as referenced to sequenced ØX174 DNA. DNA from each of the plasmids was inserted into a lambda vector DNA.
Hybrid phage containing inserted DNA of the desired size were enriched by genetic selection and their DNAs analysed by rapid
techniques. All three plasmids share the same organization, two unique sequences separated by two inverted repeats, and share
basically the same DNA sequences. Scp 2 and Scp 3 differ from Scp 1 by missing a unique Hpal site and by having small overlapping deletions in the same region. The Hpal site in Scp 1 is, therefore, in a nonessential region and suitable for insertion of foreign DNA in the potential use of
the yeast plasmid as a vector. Hybridization of labelled cloned plasmid DNA to restriction fragments of linear yeast DNA separated
on agarose gels showed that the plasmid DNA was not stably integrated into the yeast chromosomal DNA.
Recombinant plasmids were made containing cDNAs synthesized on hamster mRNAs coding for cytoskeletal (beta- or gamma-) actins and for vimentin. Hybridization of the actin probe on restriction digests of one avian and five mammalian DNAs yielded multiple bands; the vimentin probe revealed only one band (accompanied by 2-3 faint bands in some DNAs). The results obtained with the vimentin probe indicate that the corresponding coding sequences: (a) are highly conserved in warm-blooded vertebrates like the actin sequences; (b) have strongly diverged from those coding for other intermediate filament proteins, since hybridization of the vimentin probe does not lead to a diagnostic multiband pattern; and (c) most likely contribute to single gene, in contrast to the sequences coding for other cytoskeletal proteins. Hybridization of the probes on mRNAs from the different sources used showed that the non-coding sequences of both vimentin and actin genes are conserved in length.
Cloned cDNA sequences specific for alpha or beta tubulin mRNAs have been used to show that the multigene families which encode either alpha or beta tubulin are unlinked and dispersed throughout the chicken genome. Fractions of chicken chromosomes partially purified by centrifugation on a sucrose gradient were digested with restriction endonucleases and electrophoresed on agarose gels. The DNA was transferred to nitrocellulose filters and hybridized to labeled probes constructed from cloned cDNA sequences specific for alpha or beta tubulin. We find alpha tubulin sequences on four different chicken chromosomes and beta tubulin sequences on at least two different chromosomes. Moreover, using chicken chromosomes further purified with a fluorescent cell sorter, we have been able unambiguously to localize alpha tubulin genes to chromosome 1 and chromosome 8 and two of the beta genes to chromosome 2.
mRNA coding for mouse major transplantation antigens of the d haplotype was partially purified, copied into double-stranded cDNA, and cloned in Escherichia coli. Clones were selected by their ability to hybridize specifically with mRNA coding for H-2K, D, or L antigens. One of these clones, pH-2d-1, carries a 1200-base-pair insert, comprising the noncoding region, including poly(A) at the 3' end and part of the coding region. A partial sequence of the latter region showed extensive homology with the known amino acid sequences of H-2Kb,Kk, and HLA-B7 antigens. From this comparison, it appears that the coding region extends from amino acid 133 in the second domain, through the third domain, to the cytoplasmic COOH-terminal region. A stretch of 24 hydrophobic or uncharged residues, located 31 amino acids from the COOH-terminal end, could represent the segment that spans the membrane. This is followed on the cytoplasmic side of the membrane by a cluster of basic amino acids and a possible phosphorylation site on a threonine residue.
We have utilized cloned actin genes from Drosophila melanogaster and from chicken to isolate 12 actin gene fragments from a human DNA library. Each of these 12 clones was shown to contain actin coding regions by its ability to selectively hybridize to human actin mRNA as assayed by in vitro translation. The translation product was judged to be actin on the basis of its comigration with authentic actins when electrophoresed on one- and two-dimensional NaDodSO4/polyacrylamide gels and on the basis of its partial proteolysis products. Determination of the sizes and order of the fragments generated by restriction endonuclease digestion of each of these recombinant phages allows us to conclude that they are nonallelic and are from nonoverlapping regions of the genome. We have used these cloned human actin genes and the Drosophila and chicken actin gene clones to show that the human genome contains 25-30 EcoRI fragments homologous to actin genes and that, among three nonconsanguineous individuals tested, none of these fragments exhibit length or restriction-site polymorphism.
By three different lines of evidence there are approximately 20 copies of actin genes in the human genome. Firstly, the rate
of hybridisation of a mouse actin probe to human DNA indicates that there are a minimum of 20 complementary copies of the
actin sequence per genome. Secondly, this probe hybridises to 17–20 bands in Southern blots of restriction enzyme digests
of total human DNA. Most of these bands hybridise with both 3′ and 5′ fragments of the cDNA and are therefore likely to contain
the entire gene sequence. Thirdly, we have picked 12 actin recombinants from a genomic library, and at the level of restriction
enzyme mapping these represent nine different genes. Probability calculations indicate that these recombinants were picked
from a pool of at least 20 different genes.
We have investigated that organization and the distribution of a family of interspersed DNA repeats in the mouse genome. The repeats are at least 5600 base pairs (bp) in size and contain two contiguous BamHI endonuclease fragments, 4000 and 540 bp in size, the larger of which includes a 1350-bp EcoRI fragment studied by previous authors. The repeats are polymorphic in their restriction maps, and represent the major family of interspersed repeats in the mouse genome. The repeats are present almost exclusively in the two light major components of mouse DNA, and the base composition of their large BamHI fragments matches that of those components. The genomic distribution of the repeats is different from that of structural genes, which are present not only in the two light components but also in the two heavy components of mouse DNA. This distribution indicates that the repeats are not involved, at least in any simple way, in the regulation of gene expression.
A hamster actin cDNA probe was used to localize actin genes on the major components of mouse and human DNAs, namely on the four families of fragments forming the bulk of these genomes. Over 20 EcoRI fragments hybridizing the probe could be detected; a different subset of these fragments was found in each component. Since the fragment families forming the major components of the mouse and human DNAs derive from very long chromosomal segments, the isochores , the presence of actin genes on all components provides evidence for their dispersion in both genomes. In situ hybridization of 125I-labeled probe to metaphase chromosomes in the presence of dextran sulfate confirmed this dispersion by showing that the 29-30 actin gene sites so identified are distributed on almost all chromosomes. Moreover, some human actin genes could be mapped on specific chromosomal segments; in particular, one gene was localized on the long arm of the X chromosome. Finally, three different mouse actin genes were isolated from a recombinant DNA library and previously investigated interspersed repeated sequences were identified in the vicinity of these genes.
Processed genes--genes that resemble processed RNA transcripts rather than interrupted genomic sequences--have been identified as dispersed members of several gene families. Here we describe a processed gene that is one of the three human IgE-like sequences present in the human genome. The processed IgE gene has precisely lost its three intervening sequences, thereby fusing its four coding domains. The homology of the gene to its functional counterpart ends in an adenine-rich tail followed by an 11-base-pair sequence that is directly repeated 150 base pairs 5' to its first coding domain. In addition, the processed gene is located on human chromosome 9 rather than on chromosome 14, the site of the active immunoglobulin locus. The structure and evident mobility of this sequence support the concept that sequences can move about in the genome via RNA intermediates and that processed genes are a prominent feature of genomic structure.
From a human genomic library we have isolated and sequenced a β-actin-related pseudogene (HBAc-φ1) which is free of intervening sequences. Several nucleotide insertions and deletions and translational stop codons generated
within the protein-coding region indicate that this gene is functionless.
Recent years have seen rapid growth in amino acid sequence data on globins and nucleotide sequence data on haemoglobin genes and pseudogenes, and cladistic analysis1 of these data continues to reveal new facets of globin evolution. Our present findings demonstrate: (1) avian and mammalian embryonic α genes (π and ξ, respectively) had a monophyletic origin involving an α locus duplication about 400 Myr ago soon after the duplication which separated α and β genes; (2) much later in phylogeny, independent β-gene duplications produced the embryonic ρ locus of birds and embryonic ε and fetal γ loci of mammals. This parallels the earlier finding2 that myoglobins evolved more than once from generalized globin ancestors. Here we support the view2 that such globin evolution resulted from natural selection acting on mutations in duplicated genes. Thus, our evidence contradicts the neutralist view3,4 in which almost all amino acid substitutions in descent to extant globins evaded positive selection.
The sequence of two human beta-tubulin pseudogenes is described. One contains an intervening sequence but lacks sequences encoding the 55 N-terminal amino acids of the polypeptide chain. A second has no introns but has a polyadenylate signal and an oligoadenylate tract at its 3' end, and it is flanked by a short direct repeat. These sequences have arisen by different mechanisms, including one that probably involves reverse transcription of a processed messenger RNA and reintegration of the complementary DNA copy into the genome.
The urine alpha-fetoprotein (AFP) and serum albumin genes most probably arose in evolution as the consequence of a duplication of a common ancestral gene. They have both been previously mapped to chromosome 5 in the mouse. We now have evidence that these genes are closely linked. By using a unique copy DNA probe derived from previously cloned AFP 5' flanking DNA, a recombinant DNA phage has been isolated, from a bacteriophage DNA library, that contains sequences flanking the 5' end of the AFP gene and the 3' end of the albumin gene. Restriction endonuclease mapping and DNA sequence determination of the recombinant phage and comparison to total genomic DNA confirmed that the genes are in tandem, 13.5 kilobase pairs apart, with the albumin gene to the 5' side of the AFP gene. Thus, they are transcribed from the same strand of DNA.
This chapter discusses the process of detection of specific ribonucleic acids (RNAs) or specific fragments of deoxyribonucleic acid (DNA) by fractionation in gels and transfer to diazobenzyloxymethyl (DBM) paper. Small, double-stranded fragments of DNA (30–1800 base pairs) are separated with high resolution on composite gels of agarose and polyacrylamide cross-linked with N,N'-diallyltartardiamide instead of N,N′-methylenebisacrylamide. Following electrophoresis, the cleavage of the crosslinks with periodic acid facilitates the transfer of the denatured fragments to DBM paper. Detection is accomplished with labeled probes. Large fragments of DNA can also be transferred from agarose gels to DBM paper, following partial depurination with dilute acid and strand cleavage with NaOH. The efficiency of transfer is very high and independent of the size of a fragment as covalent linkage allows convenient multiple reuse of the transfers and also allows washings to be done repeatedly under stringent conditions, thereby helping to achieve very low backgrounds. Although many kinds of filter paper can be used, Whatman 540 paper was used in the study described in the chapter because of its excellent mechanical strength and resistance to chemicals. Schleicher and Schuell 589 WH paper also gives good results.
Analysis at 20 genetic loci by starch gel electrophoresis of a population from Greece shows that contrary to most accepted views, the outdoor Eastern Mediterranean Short-tailed Mouse (i) does not belong to the same species as the House Mouse (Mus musculus), (ii) is not less distant from the Eastern European semi-species of House Mouse (biochemical group 2) than from the Western and Mediterranean European semi-species (group 1), and (iii) is not more closely related to the outdoor Western Mediterranean Short-tailed Mouse Mus spretus (group 3), than to M. musculus. It represents a new biochemical group (group 4) and deserves a species rank (Mus spicilegus).
A rapid, direct method for screening single plaques of Agt recombinant phage is described. The method allows at least 10(6) clones to be screened per day and simplifies physical containment of recombinants.
We have constructed a physical map of restriction endonuclease cleavage sites in the (delta (+) beta)-globin gene region in the DNA of patients with (delta beta(0))-thalassaemia. This map shows that a 10 kb deletion has occured in (delta beta (0))-thalassaemia to remove the entire beta-globin gene and the 3' portion of the delta-globin gene. The 5' terminus of the deletion is in the large intron of the delta-globin gene and the 3' terminus 1.8 kb to the 3'-side of the beta-globin gene. A similar deletion of about 7 kb has been described previously in the DNA of patients with Hb Lepore; the 5' terminus of the deletion is also in the delta-globin gene but the 3' terminus is in the beta-globin gene. Comparison of the foetal (gamma) globin gene expression in adults with (delta beta(0))-thalassaemia and Hba Lepore suggests that the 3' extragenic regions of the beta-globin gene contain DNA sequences involved in the regulation of gamma-globulin gene expression.
Complete amino acid sequences for four mammalian muscle actins are reported: bovine skeletal muscle actin, bovine cardiac actin, the major component of bovine aorta actin, and rabbit slow skeletal muscle actin. The number of different actins in a higher mammal for which full amino acid sequences are now available is therefore increased from two to five. Screening of different smooth muscle tissues revealed in addition to the aorta type actin a second smooth muscle actin, which appears very similar if not identical to chicken gizzard actin. Since the sequence of chicken gizzard actin is known, six different actins are presently characterized in a higher mammal.
The two smooth muscle actins—bovine aorta actin and chicken gizzard actin—differ by only three amino acid substitutions, all located in the amino-terminal end. In the rest of their sequences both smooth muscle actins share the same four amino acid substitutions, which distinguish them from skeletal muscle actin. Cardiac muscle actin differs from skeletal muscle actin by only four amino acid exchanges. No amino acid substitutions were found when actins from rabbit fast and slow skeletal muscle were compared.
In addition we summarize the amino acid substitution patterns of the six different mammalian actins and discuss their tissue specificity. The results show a very close relationship between the four muscle actins in comparison to the nonmuscle actins. The amino substitution patterns indicate that skeletal muscle actin is the highest differentiated actin form, whereas smooth muscle actins show a noticeably closer relation to nonmuscle actins. By these criteria cardiac muscle actin lies between skeletal muscle actin and smooth muscle actins.
The 5S DNA of Xenopus laevis, coding for oocyte-type 5S RNA, consists of many copies of a tandemly repeated unit of about 700 base pairs. Each unit contains a "pseudogene" in addition to the gene. The pseudogene has been partly sequenced and appears to be an almost perfect repeat of 101 residues of the gene. The order of components in the repeat unit is (5') long spacer--gene--linker--pseudogene (3') in the "+" strand (or H strand) of the DNA. The possible function of the pseudogene is discussed.
— Actin present in whole rat brain cytoplasm and in synaptosomes was purified by DNase I affinity chromatography. By use of two-dimensional gels and one-dimensional isoelectric focusing gels, brain actin was shown to be composed of two isomeric forms. By comparison with muscle actins, brain actins were identified as the β and γ isomers. Muscle type α actin is not present in brain. Synaptosomal protein with high affinity for DNase I is primarily composed of β and γ actin, however, two minor synaptosomal proteins, S1 and S2, with similar DNase I affinity were also isolated. S11 and S2 have the same apparent molecular weight as whole brain actin, are more acidic than the major actin forms and are distinct from a actin. Relative to β and γ actin, the content of S1 and S2 is 3-fOld greater in synaptosomes when compared to similar non-synaptosomal species. The results demonstrate heterogeneity of brain actins and compartmentalization of brain proteins with high affinity for DNase I at the synapse. It was also shown that tubulin has selective affinity for the DNase I-actin complex.
Plasmid M6 has been shown to contain sequences complementary to two related abundant mRNA species which differ in length by 100 nucleotides and code for Dictyostelium actin. M6 complementary RNA was isolated by hybridization to immobilized M6 DNA and translated in vitro. The product is identical to major forms of in vivo labeled actin in both mobility on two-dimensional gels and two-dimensional fingerprints of tryptic peptides. Both plasmid M6 and a second plasmid complementary to the actin mRNA complementary region in M6, pDd actin 2 (McKeown et al., 1978), direct the synthesis in minicells of a number of similar polypeptides that are not seen in minicells containing other recombinant plasmids. Three of these polypeptides are similar in two-dimensional gel mobility to Dictyostelium actin and bind to DNAse I agarose. The repetition frequency of isolated restriction fragments from actin mRNA complementary plasmid M6 has been examined. The data from two different experimental approaches (DNA excess hybridizations using plasmid DNA as probe, and hybridization of plasmid probe to DNA blot filters of restriction enzyme-digested Dictyostelium DNA) indicate that the mRNA complementary region is reiterated 15--20 times. When an actin cDNA probe is used in the same experiments, the results suggest that the entire coding region is reiterated. When the two major actin mRNA species are separated and independently translated, each appears to code for one of the two major actin species. The results suggest that there are at least two different functional genes, and possibly more, for Dictyostelium actin.
Circular (e.g. simian virus 40) and linear (e.g. λ phage) DNAs have been labeled to high specific radioactivities (>108 cts/min per μg) in vitro using deoxynucleoside [α-32P]triphosphates (100 to 250 Ci/mmol) as substrates and the nick translation activity of Escherichia coli DNA polymerase I. The reaction product yields single-stranded fragments about 400 nucleotides long following denaturation. Because restriction fragments derived from different regions of the nick-translated DNA have nearly the same specific radioactivity (cts/min per 10[su3] bases), we infer that nicks are introduced, and nick translation is initiated, with equal probability within all internal regions of the DNA. Such labeled DNAs (and restriction endonuclease fragments derived from them) are useful probes for detecting rare homologous sequences by in situ hybridization and reassociation kinetic analysis.
Rabbit liver DNA was cleaved with the restriction endonucleases Eco RI, Pst I or Kpn I. Resulting DNA fragments were denatured, separated by electrophoresis in agarose gels and subsequently transferred by blotting onto nitrocellulose filters. Filters were then hybridized with a plasmid containing a DNA copy of rabbit beta-globin messenger RNA (plasmid PbetaG1; Maniatis et al., 1976) which had been labeled by nick translation to a high specific activity with 32P. A very limited number of discrete rabbit DNA fragments was found which could form well matched hybrids with PbetaG1 DNA. Endonuclease Eco RI, which cleaves the rabbit beta-globin gene, produced two beta-globin DNA fragments, whereas endonucleases Pst I and Kpn I, which do not cut the gene, each generated only one fragment. Using double digests, these fragments could be ordered into a physical map of restriction endonuclease cleavage sites around the beta-globin gene on the rabbit genome. This map is consistent with the presence of a single copy of the beta-globin gene per haploid genome. The direction of transcription of the beta-globin gene within the physical map was determined.
This paper describes a method of transferring fragments of DNA from agarose gels to cellulose nitrate filters. The fragments can then be hybridized to radioactive RNA and hybrids detected by radioautography or fluorography. The method is illustrated by analyses of restriction fragments complementary to ribosomal RNAs from Escherichia coli and Xenopus laevis, and from several mammals.
Analysis of cloned human genomic loci homologous to the small nuclear RNA U1 established that such sequences are abundant and dispersed in the human genome and that only a fraction represent bona fide genes. The majority of genomic loci bear defective gene copies, or pseudogenes, which contain scattered base mismatches and in some cases lack the sequence corresponding to the 3' end of U1 RNA. Although all of the U1 genes examined to date are flanked by essentially identical sequences and therefore appear to comprise a single multigene family, we present evidence for the existence of at least three structurally distinct classes of U1 pseudogenes. Class I pseudogenes had considerable flanking sequence homology with the U1 gene family and were probably derived from it by a DNA-mediated event such as gene duplication. In contrast, the U1 sequence in class II and III U1 pseudogenes was flanked by single-copy genomic sequences completely unrelated to those flanking the U1 gene family; in addition, short direct repeats flanked the class III but not the class II pseudogenes. We therefore propose that both class II and III U1 pseudogenes were generated by an RNA-mediated mechanism involving the insertion of U1 sequence information into a new chromosomal locus. We also noted that two other types of repetitive DNA sequences in eucaryotes, the Alu family in vertebrates and the ribosomal DNA insertions in Drosophila, bore a striking structural resemblance to the classes of U1 pseudogenes described here and may have been created by an RNA-mediated insertion event.
The genes encoding human embryonic (epsilon), fetal (G gamma, A gamma) and adult (delta, beta) beta-like globin polypeptides were isolated as a set of overlapping cloned DNA fragments from bacteriophage lambda libraries of high molecular weight (15-20 kb) chromosomal DNA. The 65 kb of DNA represented in these overlapping clones contains the genes for all five beta-like polypeptides, including the embryonic epsilon-globin gene, for which the chromosomal location was previously unknown. All five genes are transcribed from the same DNA strand and are arranged in the order 5'-epsilon-(13.3 kb)-G gamma-(3.5 kb)-A gamma-(13.9 kb)-delta-(5.4 kb)-beta-3'. Thus the genes are positioned on the chromosome in the order of their expression during development. In addition to the five known beta-like globin genes, we have detected two other beta-like globin sequences which do not correspond to known polypeptides. One of these sequences has been mapped to the A gamma-delta intergenic region while the other is located 6-9 kb 5' to the epsilon gene. Cross hybridization experiments between the intergenic sequences of the gene cluster have revealed a nonglobin repeat sequence (*) which is interspersed with the globin genes in the following manner: 5'-**epsilon-*G gamma-A gamma*-**delta-beta*-3'. Fine structure mapping of the region located 5' to the delta-globin gene revealed two repeats with a maximum size of 400 bp, which are separated by approximately 700 bp of DNA not repeated within the cluster. Preliminary experiments indicate that this repeat family is also repeated many times in the human genome.
The three active alpha-globin genes of the mouse are physically linked at a locus little more than 25 kilobases long. These genes form a part of an unexpectedly large and dispersed multigene family, including at least two pseudogenes located on two different chromosomes. This finding suggests that related gene sequences can be dispersed from their primary loci and that their dispersion may be a critical factor in the evolution of higher organisms.
We have initiated a study of the organization and expression of the actin genes of D. melanogaster. Using actin gene-specific probes from both chicken and Dictyostelium sources, a clone--denoted lambda DmA2--containing a Drosophila actin gene has been isolated from a representative library of Drosophila genomic DNA cloned in the lambda bacteriophage vector, Charon 4. Southern blotting experiments reveal that there is only one actin structural gene contained in the 17.5 kb Drosophila insert of lambda DmA2 and that the sequences immediately flanking the structural gene are single copy. Observations by electron microscopy of the R loop structures formed by hybridizing total cytoplasmic poly(A)+ RNA from Drosophila embryos to an appropriate subcloned segment of lambda DmA2 indicate that the gene consists of an approximately 70-170 nucleotide leader sequence encoding the 5' portion of the mature mRNA, a 1.65 kb intervening sequence not present in the mRNA and a 1.55 kb sequence containing the major portion of the gene. Using genomic blots with actin-specific probes derived from lambda DmA2, we show that there are six actin genes per haploid Drosophilia genome. They direct the synthesis of three major size classes of mRNA. Using in situ hybridization, the six genes have been localized to six widely dispersed sites on the polytene chromosomes; the locus for lambda DmA2 is 5C on the X chromosome. In vitro translation of mRNA selected hybridization by a DNA segment specific to lambda DmA2 suggests that this particular gene codes for one of the cytoplasmic actin polypeptides.
In this paper we describe the isolation and characterization of a 7.2 kb D. melanogaster chromosomal DNA fragment (K1) which contains nucleotide sequences complementary to D. melanogaster actin mRNA. Plasmid K1 was identified using a Dictyostelium actin cDNA plasmid, B1, as a probe. D. melanogaster mRNA selected by hybridization with immobilized K1 DNA was translated in vitro to yield products which co-migrate with the D. melanogaster actins I, II and III in two-dimensional gel electrophoresis and bind to DNAase I agarose. A physical map localizing restriction endonuclease cleavage sites in the K1 DNA fragment and the direction of transcription is presented. The position of the coding region has been localized by hybridization with labeled B1 DNA and with labeled poly(A)-containing D. melanogaster RNA. On the basis of hybridization of labeled subfragments of plasmid K1 to restriction endonuclease-cleaved D. melanogaster embryo DNA, we conclude that the nucleotide sequence of the presumptive coding region is responsible for labeling of a pattern of multiple restriction fragments from embryo DNA. The chromosomal locus from which DNA fragment K1 is derived has been localized by in situ hybridization to two closely linked bands in the region 88F. Related DNA sequences corresponding to putative actin genes have also been mapped cytologically. These results support the hypothesis that the genes for actin in D. melanogaster are members of a closely related family of coding sequences.
Previous work has shown that the macronuclear DNA of the hypotrichous ciliate Oxytricha fallax is arranged as short achromosomal pieces, 22 to 0.5 kilobase pairs (kb) in length. Micronuclear DNA has a typical chromosomal organization. Macronuclear DNA is derived from micronuclear DNA through a process of polytene chromosome fragmentation with a resultant decrease in DNA sequence complexity. Three putative actin genes have been identified in macronuclear DNA by using a cloned yeast actin gene as a hybridization probe. A restriction fragment of the yeast gene containing both actin coding and noncoding DNA hybridizes strongly to two macronuclear DNA pieces, 1.6 and 1.4 kb in length, and weakly to a 1.2-kb piece. The entire 1.6-kb piece has been cloned in plasmid pBR322 and the resulting recombinant plasmid has been designated pOfACT(1.6). The 1.6-kb pOfACT(1.6) insert hybridizes only to those restriction fragments of the yeast actin gene containing actin coding sequences. When hybridized to macronuclear DNA under conditions that allow the yeast probe to hybridize to all three macronuclear pieces, the pOfACT(1.6) insert hybridizes only to the 1.6-kb piece. Under less stringent conditions the insert also hybridizes to the 1.4-kb piece, but it shows no hybridization to the 1.2-kb DNA. The three macronuclear pieces homologous to the yeast actin gene thus differ in sequence and are interpreted as a related family of actin genes. Each of these pieces could accommodate an actin coding sequence, which in yeast, Dictyostelium discoideum, and Drosophila melanogaster is 1.1 kb, and an additional 0.1-0.5 kb of noncoding DNA.
The genomes of Mus musculus and other rodent species share a long conserved family of sequences that are dispersed and abundant (approx. 20,000 copies), and that have several novel features of organization and evolution. EcoR1 restriction of M. musculus DNA reveals a prominent 1350 bp† set of sequences. Two nonhomologous sequences of 850 and 500 bp, representing almost the total population of the 1350 bp repeats, were used to examine the detailed organization of the dispersed family and its surrounding sequences using a combination of restriction analysis and “Southern” hybridization. The 1350 bp sequence is contained within a longer repeating unit of approximately 3 kb that is dispersed amongst a wide variety of non-homologous and seemingly non-repetitive sequences. At some sites within the 3 kb repeat, considerable sequence heterogeneity has been found between members of the family, such that the family can be divided into largely non-overlapping subsets (or “segments”) according to the positioning of HinIII sites. Underlying the segmental organization there is a low background overlap of each segment with every other. Some but not all members of the family and its variants have been located on the X-chromosome in a Chinese hamster, M. musculus, X chromosome cell line: suggesting a wide genomic dispersion of the family. Homologous repeated sequences to the M. musculus 1350 bp repeat have been identified in species of Mus and Apodemus, with strikingly similar features of organization and dispersion. In M. spretus a 1350 bp sequence is contained within a dispersed repeat of at least 2·9 kb. However, the majority of M. spretus repeats contain an additional restriction site not present in the equivalent M. musculus array, suggesting a mechanism of widespread substitution or “conversion” of one variant by another in each genome. Apodemus sylvaticus possesses two dispersed and homologous families of 1350 bp and 1850 bp repetition, respectively, which contain sequences that have diverged from M. musculus to differing extents. A. mystacinus possesses only one family of dispersed and homologous repeats of 1850 bp. The majority of members within each Apodemus homologous family also contain characteristic variant restriction-site arrangements. The mechanisms underlying the spread of such variants within each array; the generation of segmental patterns; and the evolutionary conservation of this mouse interspersed family (MIF-1) are discussed in relation to the present knowledge of the organization and activity of other dispersed sequence families.
DNA rearrangements involving restriction fragment length polymorphism and variation in copy number were detected in the human genome by blot hybridization with a cloned segment of human DNA initially present in a cluster of Alu repeat sequences. These rearrangements involve both extrachromosomal circular duplex DNAs and integrated sequences, indicating the presence of transposable elements in human cells.
The immunoglobulin heavy chain loci of the mouse contain eight tightly linked genes that encode the constant region (C) of the immunoglobulin heavy chains (H). As no recombination occurred within the immunoglobulin CH gene loci among more than 3,000 crosses of mice1-3, the mouse CH genes were believed to be inherited as a set, designated the Igh haplotype. Recent molecular cloning experiments4-9 have demonstrated that the organization of the mouse Igh loci of BALB/c (Igha haplotype) is 5'-Cmu-(4.5 kilobases [kb])-Cdelta-(55kb)-Cgamma3-(34kb)-Cgamma1-(21 kb)-Cgamma2b-(15 kb)-Cgamma2a-(14 kb)-Cε-(12 kb)-Calpha-3'. The structural analyses of four Cgamma subclass genes have shown that they are essentially identical in terms of lengths and locations of each of the structural and intervening sequences6,10-12. Comparison of the nucleotide sequences of the Cgamma1, Cgamma2b and Cgamma2a genes has shown that limited portions of the Cgamma gene are conserved between compared pairs of the Cgamma genes12. The results imply that the nucleotide sequences of the Cgamma gene segments have been exchanged by recombination among related clustered genes. Similar studies on the Cgamma2a genes of different haplotypes, Igha and Ighb, have also suggested that these two types of Cgamma2a gene have undergone recombinational exchange of a part of the gene sequence13. These results imply, in contrast to the previous belief, that frequent recombinational events have taken place within the Igh loci and that their nucleotide sequences have been rearranged during evolution. Here we present direct evidence that a Japanese wild mouse, Mus musculus molossinus, contains duplicated Cgamma2a genes while the copy numbers of the neighbouring CH genes, that is, the Cgamma1, Cgamma2b and Cε genes, remain constant. The results indicate that the duplicated Cgamma2a genes of M. m. molossinus arose from unequal crossing-over between homologous chromosomes.
Screening of two human genomic libraries with a chicken α-tubulin complementary DNA probe has resulted in the isolation of several clones containing a common hybridizing region. Restriction mapping of the cloned fragments reveals that, while the majority of sites are retained in each clone, there are a number of differences, including several within the gene-containing region. These differences cannot be ascribed solely to restriction-site polymorphism, and therefore reflect the existence of a family of closely related α-tubulin genes.
The term pseudogene was coined by Jacq et al. to describe 'genes' within the 5S cluster of Xenopus laevis, which, while they are clearly related to normal 5S genes, are incapable of producing functional 5S RNA sequences. The last 5 years have seen the appearance of pseudogenes in many gene families, but it is among the globin genes that the broadest repertoire of pseudogene structure and dysfunction is observed. As a working definition, a globin pseudogene is a DNA sequence that is originally derived from a functional globin gene, that is not capable of producing functional globin protein and that is apparently fixed in the population. Functional homologs of the pseudogene must ordinarily be present. This does not exclude the possibility of expression of nonglobin proteins encoded by globin pseudogenes. Sequences that correspond to this definition have been found in humans, goats, mice and rabbits, and their presence is inferred in several monkey species. Now are pseudogenes generated? Duplication of different regions of DNA followed by sequence divergence appears to be a plausible answer. The work of several different laboratories has suggested that globin loci evolve by a series of gene duplications. Shen et al. indicate that following the duplication of the γ-globin genes in humans, exchange of information between the duplicated genes on the same DNA strand occurred, probably by recombination and subsequent gene conversion. It is not known how paired genes break free of this correction mechanism. Presumably, this will depend on the relative rates of mutation and correction. Accumulation of clustered mutations and insertions or deletions might be expected to decrease correction rates. Why do pseudogenes spread through the population? There are two classical explanations for gene fixation in populations: fixation through selection and through drift. Rates of divergence of pseudogenes from their functional counterparts are comparable with those seen in noncoding and presumably unselected sequences, and this indicates that pseudogenes do not have a sequence-specific function. Altogether it is probably most attractive to see pseudogenes as analogous to neutral mutations, fixed in the population by drift or occasionally hitch-hiking with linked sequences that are under selection.
The nucleotide sequence of the chick a-actin gene reveals that the gene is comprised of 7 exons separated by six very short
intervening sequences (IVS). The first IVS interrupts the 73 nucleotide 5′ untranslated segment between nucleotides 6l and
62. The remaining IVS interrupt the translated region at codons 41/42, 150, 204, 267, and 327/328. The 272 nucleotide 3′ untranslated
segment is not interrupted by IVS. The amino acid sequence derived from the nucleotide sequence is identical to the published
sequence for chick a-actin except for the presence of a met-cys dipeptide at the amino-terminus. The IVS positions in the
chick a-actin gene are identical to those of the rat a-actin gene. While there is partial coincidence of the IVS positions
in the a-actin genes with the vertebrate b-actin genes and 2 sea urchin actin genes, there is no coincidence with actin genes
from any other source except soybean where one IVS position is shared. This discordance in IVS positions makes the actin gene
family unique among the eucaryotic genes analyzed to date.
The actins constitute a family of highly conserved proteins found in all eukaryotic cells. Their conservation through a very wide range of taxonomic groups and the existence of tissue-specific isoforms make the actin genes very interesting for the study of the evolution of genes and their controlling elements. On the basis of amino acid sequence data, at least six different mammalian actins have been identified (skeletal muscle, cardiac muscle, two smooth muscle actins and the cytoplasmic beta- and gamma-actins). Rat spleen DNA digested by the EcoRI restriction enzyme contains at least 12 different fragments with actin-like sequences but only one which hybridized, in very stringent conditions, with the skeletal muscle cloned cDNA probe. Here we describe the sequence of the actin gene in that fragment. The nucleotide sequence codes for two amino acids, Met-Cys, preceding the known N-terminal Asp of the mature protein. There are five small introns in the coding region and a large intron in the 5'-untranslated region. Comparison of the structure of the rat skeletal muscle actin gene with available data on actin genes from other organisms shows that while the sequenced actin genes from Drosophila and yeast have introns at different locations, introns located at codons specifying amino acids 41, 121, 204 and 267 have been preserved at least from the echinoderm to the vertebrates. A similar analysis has been done by Davidson. An intron at codon 150 is common to a plant actin gene and the skeletal muscle acting gene.
We describe ten Charon 4A genomic DNA clones from BALB/c mice which include at least seven different major urinary protein (MUP) genes. We have established the orientation of all seven sequences, and have placed six of them in precise register by means of restriction site maps and Southern blot hybridization with cloned cDNA sequences. Four of the seven genomic sequences (family I sequences) form hybrids with six independent cDNA clones that have a high thermal stability and hybridize more strongly with mRNA from three inbred mouse lines. Hybrids between the remaining three genomic sequences and the cDNA clones have a lower thermal stability and hybridize less strongly with mRNA from the three inbred lines. Homologies between different cloned sequences extend over as much as 15 kb. No clone contains parts of two MUP genes, and no homology has been detected between the 3' flanking region of one MUP gene and the 5' flanking region of another.
The white-ivory (wi) mutation, an unstable allele of the white locus in Drosophila, reverts to wild-type at frequencies of 5 X 10(-5) in homozygous females, and 5 X 10(-6) in males and deletion heterozygous females. We show by molecular cloning and Southern blot analysis of DNA from wi flies that a 2.9 kilobase tandem duplication within the white locus is responsible for the mutation. Phenotypic reversion appears, in most cases, to be due to an exact excision of the extra copy of the sequence. Two derivative alleles of wi, one phenotypically wild-type, the other a partial revertant, carry insertions of moderately repetitive DNA from outside the locus, in addition to suffering deletions of some white locus DNA. Earlier genetic data preclude unequal crossing-over between homologs as an explanation for the precise reversions. Rather, an intrachromosomal meiotic event seems to be responsible. Our results suggest that intrachromosomal recombination may be responsible in other systems for a larger number of rearrangements than has been suspected, and that interallelic recombination frequencies in Drosophila do not always correlate in a simple way with DNA length or extent of homology.
From Acanthamoeba castellanii we have isolated and completely sequenced an actin gene which is interrupted by a 129 base-pair intervening sequence following the codon for amino acid 105. S1 nuclease mapping experiments indicated that this gene (actin gene I) is expressed in vivo. There is only one size class (1300 nucleotides) of actin messenger RNA in Acanthamoeba. We have used specific fragments of the isolated gene to demonstrate that there are at least three and possibly more actin genes in this organism. As deduced from the DNA structure, the Acanthamoeba actin I is a protein of 374 amino acids and has an unusual glycine residue at its N-terminus.
The genormic organization of the multiple actin DNA sequences in the lower eukaryote Physarum polycephalum was investigated by Mendelian mapping. Actin-homologous restriction endonuclease cleavage fragments detected by DNA blotting showed length polymorphisms when different strains were compared. These length polymorphisms were used as phenotypic markers for actin sequences in the genome. The meiotic assortment of the polymorphic restriction fragments was analysed, revealing four unlinked actin loci. The data for three-of the actin loci, ardB, C and D, are consistent with a single sequence or gene at each locus. The data for the other actin locus. ardA, is consistent with multiple linked actin sequences or genes.
Four actin genes have been isolated from Caenorhabditis elegans that account for all of the major actin hybridization to total genomic DNA. Actin genes I, II and III are clustered within a 12 X 10(3) base region; gene IV is unlinked to the others. All four genes have been sequenced from at least nucleotide -109 to +250. Genes I and III are identical for the first 307 coding nucleotides. Genes I and II differ in 14 positions within the first 250 coding nucleotides; one difference substitutes an aspartic acid for a glutamic acid at codon 5. Genes I and IV differ in 18 positions within the first 259 coding nucleotides without causing any amino acid differences. Genes I, II and III have introns after the first nucleotide of codon 64 and gene IV has an intron between codons 19 and 20. The four nucleotide sequences thus far define two different amino acid sequences. Both of the amino acid sequences resemble vertebrate cytoplasmic actin more than vertebrate muscle actin. A DNA polymorphism between the Bristol and Bergerac strains has been used as a phenotypic marker in genetic crosses to map the cluster of actin genes within a 2% recombination interval on linkage group V between unc-23 and sma-1 in order to begin a molecular genetic analysis of the actin loci.
Two recombinant phages that contain cardiac muscle actin gene were isolated from a human DNA library and their structures were determined. Restriction analysis indicates that both clones carry the same EcoRI 13-kilobase fragment where the coding sequence is mapped. The cloned DNA hybridized with polyadenylylated RNA from human fibroblasts, which directs the synthesis of cytoplasmic beta- and gamma-actin in vitro. However, sequence determination of the cloned DNA showed that the entire coding sequence perfectly matched the amino acid sequence of cardiac muscle actin. The initiation codon is followed by a cysteine codon that is not found at the amino-terminal site of any actin isoform, suggesting the necessity of post-translational processing for in vivo actin synthesis. There are five introns interrupting exons at codons 41/42, 150, 204, 267, and 327/328. Surprisingly, these intron locations are exactly the same as those of the rat skeletal muscle actin gene but different from those of nonmuscle beta-actin gene. Nucleotide sequences of all exon/intron boundaries agree with the G-T/A-G rule (G-T at the 5' and A-G at the 3' termini of each intron). The 3'-untranslated sequence has no homology to that of nonmuscle beta- or gamma-actin gene, but Southern blot hybridization has shown that this region has considerable homology to that of one of the other actin genes. These results indicate that the recombinant phages, which we have isolated, contain cardiac muscle actin gene and that cardiac muscle actin gene and skeletal muscle actin genes are derived from their ancestor gene at a relatively recent time in evolutionary development.
The events of B-cell differentiation can be reconstructed in part through an analysis of the organisation of heavy-chain gene segments in differentiated B cells. A mouse immunoglobulin alpha heavy-chain gene is composed of at least three noncontiguous germ-line DNA segments--a VH gene segment, a JH gene segment associated with the Cmu gene segment, and the C alpha gene segment. These gene segments are joined together by two distinct types of DNA rearrangements--a V-J joining and a CH switch.
Southern transfer and solution hybridization experiments, using as probe a DNA fragment that encodes for Drosophila actin, demonstrate cross hybridization to DNA from the sea urchin Strongylocentrotus purpuratus. Recombinant DNA clones that contained sea urchin genomic DNA fragments were constructed and screened for the presence of actin-encoding DNA sequences by colony hybridization with the Drosophila actin sequence. Two different putative actin-encoding clones were identified and were shown to specifically hybridize actin-encoding mRNA from a complex mRNA population. Southern blot hybridization experiments with both the Drosophila actin sequence and one of the cloned sea urchin sequences, in conjunction with solution hybridization data, suggest an actin gene copy number of 5-20 per haploid genome. DNA sequence analysis of one of the cloned sequences indicates that this fragment codes for a cytoplasmic form of actin and contains an intervening sequence of at least 200 nucleotides beginning immediately after amino acid 121 in the protein sequence.
Based on the finding that the amino-terminal tryptic peptide of actin is a reliable marker for actin divergence, we describe in detail a highly sensitive protein-chemical procedure for actin typing. The method is performed on non-radioactivity labeled cells and tissues and six actins can be identified unambiguously in warm-blooded vertebrates. The method is quantitative and gives directly the ratio of the different actions in the specimens. It does not require previous purification of actin and can be used on total cellular extracts without any prior fractionation. The procedure can be extended to actins not previously characterized by amino acid sequence analysis and makes certain predictions possible about the partial amino acid sequences of the amino-terminal tryptic peptides, mostly sufficient for a correlation with DNA sequences derived from cloned actin genes. This is done as an example for the cytoplasmic action present in Schneider L-2 Drosophila melanogaster cells. Although the method is currently used routinely on 10(5) cells, modifications are discussed, which should allow the analysis to be performed with even higher sensitivity.
We have discovered in animal cells a novel class of dispersed, solitary genetic elements derived from tandem multigene families. We refer to such displaced elements as orphons. Orphons arise from both protein-coding and non-protein-coding structural gene families, including those of histone and ribosomal genes. Southern transfer hybridization experiments, on DNA digested with enzymes that do not cut the major repeat unit of the family of genes of interest, reveal histone gene orphons in the sea urchin (Lytechinus pictus), ribosomal gene orphons in yeast (Saccharomyces cerevisiae) and ribosomal and H3 histone gene orphons in Drosophila melanogaster. There are more than 50 histone gene orphons in each sea urchin genome. Each of the five histone-coding regions has a number of orphons (5--20), in addition to the several hundred copies in the clusters that are not cut by Bam HI. Most such orphons appear to contain only one coding region. Nearly all sea urchin histone gene orphon loci are polymorphic in the population; no two individuals have the same sets of histone orphons for any coding region. An H3-coding orphon, pLpH30-1, was isolated from the genome of L. pictus. The H3 region homologous to the histone gene clusters is 1200 bases long and includes the complete early H3 gene and some surrounding histone DNA. THe orphon is flanked by at least 1.7 kb of 5' and 3 kb of 3' nonhistone DNA. These flanking sequences are each moderately repetitive in the genome and are not homologous to each other. The orphon differs less than 2% in base sequence from the analogous region of pLpC and only slightly more from pLpA (the major histone gene clones). DNA sequence analysis of the junctions between the H3 region and the immediate flanking nonhistone DNA reveals no evidence of repetitive or palindromic sequences.
A dispersed immunoglobulin pseudogene carries two hallmarks of RNA
processing-spliced J and C regions and a poly (A)-rich tail. Its
discovery strengthens the notion that processed genes are a significant
feature of the mammalian genome and that genetic information can return
to the genome via an RNA intermediate.