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The actin genes of Drosophila: protein coding regions are highly conserved but intron positions are not
Abstract
The entire set of six closely related Drosophila actin genes was isolated using recombinant DNA methodology, and the structures of the respective coding regions were characterized by gene mapping techniques and by nucleotide sequencing of selected portions. Structural comparisons of these genes have resulted in several unexpected findings. Most striking is the nonconservation of the positions of intervening sequences within the protein-encoding regions of these genes. One of the Drosophila actin genes, DmA4, is split within a glycine codon at position 13; none of the remaining five genes is interrupted in the analogous position. Another gene, DmA6, is split within a glycine codon at position 307; at least two of the Drosophila actin genes are not split in the analogous position. Additionally, none of the Drosophila actin genes is split within codon four, where the yeast actin gene is interrupted. The six Drosophila actin genes encode several different proteins, but the amino acid sequence of each is similar to that of vertebrate cytoplasmic actins. None of the genes encodes a protein comparable in primary sequence to vertebrate skeletal muscle actin. Surprisingly, in each of these derived actin amino acid sequences in the initiator methionine is directly followed by a cysteine residue, which in turn precedes the string of three acidic amino acids characteristic of the amino termini of mature vertebrate cytoplasmic actins. We discuss these findings in the context of actin gene evolution and function.
Supplementary resource (1)
Nucleotide Sequence
May 1997
... However, in all published reports to date regarding protostome and deuterostome actin genes this methionine codon is always followed by a cysteine codon. This is true of all six actin genes in Drosophila melanogaster (7). It is also of interest that although all these Drosophila genes encode cytoplasmic-like actin proteins, some are expressed in muscle. ...
... Note the presence of a cysteine codon immediately after the initiator methionine codon in a-actin (boxed) but not in or y-actin mRNA. mRNA contains the consensus sequence CAA Pu AUG found at the initiation site of five of the six Drosophila actin genes (7). ...
... The problem derives from the following considerations. First, the presence of a cysteine residue at amino acid 1 (referred to as "Cys+") is unique to protostome and deuterostome actin genes (2,6,7,29), whereas all actin genes in more primitive organisms lack this residue (called "Cys-") (5,8,16). Second, the ubiquitous occurrence of both Cys+ and a mechanism to remove the aminoterminal cysteine in both protostomes and deuterostomes is too coincidental to be explained by convergent evolution. ...
cDNAclones encoding three classes ofhumanactins have been isolated and characterized. Thefirst twoclasses (yand,B, cytoplasmic actins) wereobtained froma cDNA library constructed fromsimian virus 40-transformed human fibroblast mRNA,andthethird class (a,muscle actin) wasobtained fromacDNA library constructed fromadult humanmuscle mRNA.A newapproach was developed toenrich forfull-length cDNAs.Thehumanfibroblast cDNAplasmid library waslinearized withrestriction enzymes thatdidnotcuttheinserts of interest; itwasthensize-fractionated ongels, andthechimeric molecules of
... The actins found in most invertebrate species resemble the nonmuscle actin isoforms found in higher vertebrates (12), whereas the appearance of striated actin isoforms correlates with the evolutionary transition from primitive deuterostomes to chordates (J. Vandekerckhove, Abstr. ...
... Since these assays are sensitive down to only 5% of the total actin population, it is possible that some minor actins might have escaped detection. This analysis is especially relevant to the nonmuscle actins in which a large diversity of isoforms have been found in invertebrates (12,15,23) as well as in cold-blooded vertebrates (46). For example, in a study examining nonmuscle actin isoforms in amphibians, at least 5 different nonmuscle actin isoforms were resolved (46). ...
... Dodgson et al. (8) and screened as detailed by Maniatis et al. (28). Plaques (3 x 105) were first screened with a nicktranslated HindIII fragment of Drosophila actin gene XDmA2 (12) which contains the entire coding sequence, a single intron, and 5'-and 3'-flanking sequences. After a number of plaque purification steps, 26 of 30 plaques consistently hybridized to cloned chicken a-skeletal and P-actin cDNA. ...
... Reference sequences for primer design were obtained from the literature (Konsolaki et al. 1990;Rina & Savakis 1991;He & Haymer 1992;Kwiatowski et al. 1992). The loci investigated were muscle-specific actin (Actin) intron 1, chorion s36 (s36) intron 1, vitellogenin 1 gamma (Vg1) intron 2, and Cu/Zn superoxide dismutase (SOD) Fyrberg et al. 1981;Sanchez et al. 1983;Konsolaki et al. 1990;Tolias et al. 1990;Rina & Savakis 1991;Kwiatowski et al. 1992. ...
... There are also multiple fixed differences between these three genes (i.e. Vanderkerckhove & Weber 1978;Fyrberg et al. 1981), some of which are in the exon sequences amplified using EPIC primers. Our primers targeted the muscle-specific actin. ...
Biological invasions generally start from low initial population sizes, leading to reduced genetic variation in nuclear and especially mitochondrial DNA. Consequently, genetic approaches for the study of invasion history and population structure are difficult. An extreme example is the Mediterranean fruit fly, Ceratitis capitata (Medfly), for which successive invasions during this century have resulted in a loss of 60% of ancestral genetic variation in isozymes and 75% of variation in mitochondrial DNA. Using Medflies as an example, we present a new approach to invasion genetics that measures DNA sequence variation within introns from multiple nuclear loci. These loci are so variable that even relatively recently founded Medfly populations within California and Hawaii retain ample genetic diversity. Invading populations have only lost 35% of the ancestral genetic variation. Intron variation will allow high-resolution genetic characterization of invading populations in both natural and managed systems, although non-equilibrium methods of analysis may be necessary if the genetic diversity represents sorting ancestral polymorphism.
... The C3-22 probe was a 466 bp fragment from the C3-22 coding sequence ( Penalva et al. 1997). The actin probe consisted of a 650 bp fragment containing 5 0 noncoding and coding sequences of the Drosophila actin gene ( Fyrberg et al. 1981). In vitro transcription was performed in the presence of [a-32 P]dUTP, using the Maxiscript T7/T3 kit (Ambion). ...
... The transcriptional activity of the C3-22 gene and its temporal regulation were analyzed in the transgenic lines using the ribonuclease protection method (Sambrook and Russel 2001). A 650 bp RNA probe corresponding to a portion of the actin gene of Drosophila ( Fyrberg et al. 1981), which resulted in the protection of a 260 bp fragment, was used as control for the quantity of RNA on the gel. The probe used to detect the mRNA of the C3-22 gene was an RNA of 535 bp corresponding to a portion of its cDNA, which resulted in the protection of a fragment of about 466 bp. ...
Drosophila melanogaster was transformed with an 18kb fragment of the C3 DNA puff of Rhynchosciara americana, including the C3-22 gene and the origins of replication that direct amplification. Different tissues and developmental stages of five independent transgenic lines were analyzed by quantitative Southern blot hybridization. No indication was found that the transformed fragment was amplified, strongly suggesting that factors involved in DNA puff amplification have not been conserved in Drosophila. Transcription of the C3-22 gene in the transgenic lines was found to be at a low and constitutive level throughout development. These results indicate that, unlike other DNA puff genes, the factors that regulate the C3-22 gene are not conserved in Drosophila.
... Previously published sequences were obtained from Gen-Bank/EMBL/DDBJ and used for amino acid comparison and phylogenetic analyses. The following is a list of accession numbers for the actin sequences; M20543, human α-skeletal muscle (Taylor et al. 1988); J00073, human α-cardiac muscle (Hamada et al. 1982); X13839, human α-aortic smooth muscle (Kamada and Kakunaga 1989); X16940, human γ-enteric smooth muscle (Miwa and Kamada 1990); M10277, human β-cytoplasmic (Nakajima-Iijima et al. 1985); M19283, human γ-cytoplasmic ); D10887, Halocynthia roretzi larval muscle HrMA4 (Kusakabe et al. 1992); D45164, H. roretzi cytoplasmic HrCA1 (Araki et al. 1996); D85743, Molgula oculata adult muscle MocuMA2 (Kusakabe et al. 1997); X61041, S. plicata cytoplasmic SpCA8 (Kovilur et al. 1993); M26500, Pisaster ochraceus muscle (Kowbel and Smith 1989); M26501, P. ochraceus cytoplasmic (Kowbel and Smith 1989); M18829, Drosophila melanogaster muscle 79B (Fyrberg et al. 1981); K00667, D. melanogaster cytoplasmic 5C (Fyrberg et al. 1981); X06363, Bombyx mori muscle (Mounier et al. 1987); M20016, Arabidopsis thaliana AAc1 (Nairn et al. 1988). ...
... Previously published sequences were obtained from Gen-Bank/EMBL/DDBJ and used for amino acid comparison and phylogenetic analyses. The following is a list of accession numbers for the actin sequences; M20543, human α-skeletal muscle (Taylor et al. 1988); J00073, human α-cardiac muscle (Hamada et al. 1982); X13839, human α-aortic smooth muscle (Kamada and Kakunaga 1989); X16940, human γ-enteric smooth muscle (Miwa and Kamada 1990); M10277, human β-cytoplasmic (Nakajima-Iijima et al. 1985); M19283, human γ-cytoplasmic ); D10887, Halocynthia roretzi larval muscle HrMA4 (Kusakabe et al. 1992); D45164, H. roretzi cytoplasmic HrCA1 (Araki et al. 1996); D85743, Molgula oculata adult muscle MocuMA2 (Kusakabe et al. 1997); X61041, S. plicata cytoplasmic SpCA8 (Kovilur et al. 1993); M26500, Pisaster ochraceus muscle (Kowbel and Smith 1989); M26501, P. ochraceus cytoplasmic (Kowbel and Smith 1989); M18829, Drosophila melanogaster muscle 79B (Fyrberg et al. 1981); K00667, D. melanogaster cytoplasmic 5C (Fyrberg et al. 1981); X06363, Bombyx mori muscle (Mounier et al. 1987); M20016, Arabidopsis thaliana AAc1 (Nairn et al. 1988). ...
The cephalochordate amphioxus is thought to share a common ancestor with vertebrates. To investigate the evolution of developmental
mechanisms in chordates, cDNA clones for two amphioxus actin genes, BfCA1 and BfMA1, were isolated. BfCA1 encodes a cytoplasmic actin and is expressed in a variety of tissues during embryogenesis, beginning in the dorsolateral mesendoderm
of the mid-gastrula. At the open neural plate stage, BfCA1 transcripts accumulate at the bases of the neuroectodermal cells adjacent the presumptive notochord. The 3’ untranslated region
of BfCA1 contains a sequence that is similar to the ”zipcode” sequence of chicken β-cytoplasmic actin gene, which is thought to direct
intracellular mRNA localization. BfCA1 is also expressed in the notochord through the early larval stage, in the pharynx and in the somites at the onset of muscle-cell
differentiation. BfMA1 is a vertebrate-type muscle actin gene, although the deduced amino acid sequence is fairly divergent. Transcripts first appear
in the early neurula in the somites as they begin to differentiate into axial muscle cells and persist into the adult stage.
In young adults, transcripts are localized in the Z-discs of the muscle cells. Smooth muscle cells around the gill slits and
striated muscle cells in the pterygeal muscle also express BfMA1; however, there is never any detectable expression in the notochord, which is a modified striated muscle. Together with the
alkali myosin light chain gene AmphiMLC-alk, the sequence and muscle-specific expression of BfMA1 implies a conserved mechanism of muscle cell differentiation between amphioxus and vertebrates. Evolution of the chordate
actin gene family is discussed based on molecular phylogenetic analysis and expression patterns of amphioxus actin genes.
... In this strategy, one of the four actin genes found in Ae. aegypti, actin-4, was found to be essential for female ight. This and other studies in model organisms, such as Drosophila melanogaster, have shown that the identi cation of the speci c functions of particular actin genes in each species can be complex, as they are highly conserved and can show more than 90% similarity between isoforms (18)(19)(20)(21)(22)(23). AeAct-4 and a similar orthologue have been reported as a female-biased gene in adults of Ae. aegypti and Anopheles stephensi (24,25). ...
Background
Despite the progress to eliminate malaria in Central America, focalized transmission persists, and insecticide resistance is on the rise in the primary vector, Anopheles albimanus. Many of the new control methods being developed depend on the release of a large number of male mosquitoes that must be sorted prior to release. However, An. albimanus manual pupal-sex-sorting is not feasible, and therefore, we explored the use of RNA interference (RNAi) targeting genes with a sex-biased expression for female elimination. Here, we evaluated the effect of feeding larvae with dsRNA for a female-biased orthologue of the flight muscle actin gene.
Results
Two sex-biased actin forms were identified in An. albimanus. Gene expression analysis showed a >40-fold higher expression of the AALB015469 transcript in female pupae (p = 0.0048) and adults (p = 0.0078) when compared to males. Tissue-specific analysis also suggests this female-biased actin can be an orthologue of the flight muscle actin of Aedes aegypti. At the same time, the AALB015481 transcript showed a >40-fold higher expression in male pupae and adults when compared to females, with no detectable expression in flight muscle. The potential effects of oral-induced RNAi for the female-biased actin were evaluated. Larvae were fed a diet containing either dsRNA for the female-biased actin 3'-UTR alone or for the UTR with an adjoining portion of the C-terminal coding region. A significant number of flightless females resulted from feedings with 3' UTR alone (10.50 ± 5.92 %, p < 0.05) or with the coding region (6.00 ± 2.16 %, p < 0.01). Treatment with the 3' UTR alone resulted in a significant number of flightless males (8.25 ± 3.10 %, p < 0.01). Both diets produced significant mortality in both female and male adults (p < 0.0001).
Conclusions
Feeding of An. albimanus larvae with dsRNA targeting the female-biased flight muscle actin orthologue impairs flight in both sexes and affects the overall survival of female and male mosquitoes. Providing dsRNA in the larval diet shows promise as a method for screening other differentially expressed genes as potential targets for female elimination in mosquito breeding facilities.
... This is in keeping with observations on many other genes such as the carbonic anhydrase II gene (Venta et al, 1985), the a l -antitrypsin and ovalbumin genes (Leicht etal, 1982) and the gene for amylase (MacDonald etal, 1980) where no correlation can be found between the individual exons or groups of exons and structural features of the protein. An alternative model for the positioning of introns, proposed by Fyrberg (1981), is that some introns are vestiges of transposon-like elements that have been inserted into genes and become fixed and have subsequently diverged in nucleotide sequence. ...
Phosphoglucomutase is an enzyme central to glycolysis and gluconeogenesis. There are three well characterised forms: PGM1, PGM2 and PGM3. Genomic clones containing the 5' end of the PGM1 gene were isolated and characterised. The exon/intron boundaries of the first exon (designated 1 A) were determined. 15kb of intron 1 have been mapped and 2.8kb of the flanking regions sequenced. The proximal promoter shows features of a 'housekeeping' promoter. Features characteristic of a CpG island have been identified in this region. An alternative first exon has been identified (1B). The two exons show similarities indicating a gene duplication event. Exon 1B is transcribed from a promoter in the first large intron of PGM 1.1 A. Expression studies of PGM 1.IB by RNA-PCR show a limited tissue expression, with predominate expression in striated muscle. Exons 1A and 1B were sequenced in DNA from eight individuals of known PGM1 protein phenotypes. No genetic variation in these sequences was encountered. The PGM1 cDNA was used as a probe in a search for the PGM3 gene. A human chromosome 6 library was screened. Positive cosmids were assigned to chromosome positions by fluorescent in situ hybridisation, restriction mapped and sequenced in part. These studies led to the identification of a novel PGM on chromosome 9. PGM3 was not isolated by this procedure, suggesting that the sequences of PGM1 and PGM3have diverged significantly. Mammary gland cDNA libraries were screened using both anti-PGM1 antibodies and PGM1 cDNA, to search for PGM4. Positive clones were all identified as PGM1. In addition the PGM1 type of DNA from recent mothers was compared to the typing of matched colostrum samples (by isoelectric focusing). Some similarities were seen between the colostrum and PGM1 isoform patterns. Together, these data suggest that PGM4 protein is a modified form of PGM1.
... Among these, we were able to annotate 88 genes in the Oncopeltus genome, Pediculus humanus, Anopheles gambiae and Drosophila melanogaster revealed a median of ~46 % protein identity. Drosophila alignment in particular shows that our dataset contains many genes encoding proteins recovered with full length, such is the case of the highly conserved gene Actin-5C[159]. Alignments show that many Oncopeltus genes with shorter sequence than Drosophila homologs have missing sequence primarily in the N-terminal and C-terminal. ...
... Recent phylogenetic analysis based on accurate tree-based orthology estimation has shown that despite their conserved muscular expression, chordate and non-chordate muscle actin genes are not orthologous, but they have a polyphyletic origin due to independent duplications from cytoplasmic genes in different lineages (Inoue and Satoh, 2018). This finding is consistent with previous analyses that based on comparisons of diagnostic positions and exon-intron structures had classified musclespecific genes of non-chordate animals together with the cytoplasmic actin genes (Chiba et al., 2003;Fyrberg et al., 1981;Kusakabe et al., 1997b;Vandekerckhove and Weber, 1984). ...
Locomotion by tail beating powered by a system of bilateral paraxial muscle and notochord is likely one of the key evolutionary innovations that facilitated the origin and radiation of chordates. The innovation of paraxial muscle was accompanied by gene duplications in stem chordates that gave rise to muscular actins from cytoplasmic ancestral forms, which acquired contractile capability thanks to the recruitment of the myosin motor-machinery. To better understand the role of actin diversification during the evolution of chordates, in this work we have characterized the complete actin catalogue of the appendicularian Oikopleura dioica, an urochordate that maintains a chordate body plan throughout its life, including the notochord in a muscled tail that confers an active free-living pelagic style. Our genomic survey, phylogenetic analyses and Diagnostic-Actin-Values (DAVs) reveal that O. dioica has four muscular actins (ActnM1-4) and three cytoplasmic actins (ActnC1-3), most of which originated by independent gene duplications during the evolution of the appendicularian lineage. Detailed developmental expression atlas of the complete actin catalogue of O. dioica reveals differences in the temporal-regulation and tissue-specificity of different actin paralogs, suggesting complex processes of subfunctionalization during the evolution of urochordates. Our results suggest the presence of a "cardio-paraxial" muscular actin at least in the last common ancestor of Olfactores (i.e. vertebrates+urochordates). Our results reveal highly dynamic tissue-specific expression patterns for some cytoplasmic actins, including the notochord, ciliated cells and neurons with axonal projections, which challenge the classic housekeeping notion ascribed to these genes. Considering that previous work had demonstrated the existence of notochord-specific actins in cephalochordates, the tissue-specific expression of two cytoplasmic actins in the notochord of O. dioica suggests that this pattern plausibly reflects the ancestral condition of chordates, and provides new insights to better understand the evolutionary origin of the notochord.
... The putative muscle-specific cDNA sequences of the act2a and act2b loci are 100% identical. Their deduced amino acid sequences contain residues (e.g., A233 within the tropomyosin-binding domain, G369 near the C termini) which are also conserved with, and specific for, muscle-specific actin proteins of Drosophila and lobster (Homarus americanus) (Fyrberg et al. 1981;Kim et al. 2009). There was no clear evidence of residues suggesting a heart-specific actin in D. pulex when compared to the heart-specific actin of H. americanus (Kim et al. 2009). ...
Cladocerans (water fleas) are planktonic crustaceans that typically have a bivalved carapace. Each valve of the carapace consists of two cuticle-secreting epithelial layers that are separated by a hemolymphatic chamber and joined by pillar structures. Ultrastructural analyses in several species of Cladocera have shown that the carapace epithelia and pillars contain filamentous structures of unknown composition. In the present study we used a fluorescent phalloidin conjugate to show that the carapaces of three cladocerans, Daphnia magna, D. pulex, and Sida crystallina, are rich in large bundles of filamentous actin (F-actin). In D. magna we employed confocal microscopy and orthogonal views of three-dimensional reconstructions to show that these bundles extend radially from foci in the pillars towards the integument surfaces, and their structure is consistent with that of contractile stress fibers. Using a fluorescent lipophilic stain, DiOC6(3), we show that the F-actin bundles are distributed in membrane-rich regions within the carapace epithelia, and that, in the superficial epithelium, these may be large membrane-bound organelles. In D. magna, the F-actin bundles are present in embryonic, juvenile instar, and adult, developmental stages, and through development the bundles become larger, contain more F-actin, and become more widely spaced. We present an alignment of the deduced amino acid sequences of six putative D. pulex actin genes, and discuss the implications that their respective sequences have on the likelihood of their inclusion into the F-actin bundles of the carapace. Our identification of these large F-actin bundles within the pillars of three cladocerans provides new insight into the role these structures play in influencing carapace dynamics within this order.
... Expression across all tissues indicates that four genes encode cytoplasmic actins (Fig. 3). Protein sequence comparisons placed all B. glabrata actins as most closely related to mammalian cytoplasmic rather than sarcomeric actins (Supplementary Note 30; Supplementary Data 39), a pattern also observed for all six actin genes of D. melanogaster 44 . The actin genes of B. glabrata and other molluscs were most similar to paralogs within their own genomes, rather than to other animal orthologs (Fig. 3). ...
This corrects the article DOI: 10.1038/ncomms15451.
... While both insects and mammals have actin orthologs that are expressed in distinct muscle and cell types, the evolutionary origin of these genes is interesting. In particular, all of the six Drosophila melanogaster actin genes are closest in amino acid sequence similarity to mammalian cytoplasmic actins, rather than to any of the mammalian muscle actins 297 . This and subsequent data have led to the proposal that single actin gene precursors diverged independently in arthropod versus mammalian lineages, to give rise to the families of actin genes observed today 298 . ...
Supplementary Figures, Supplementary Notes and Supplementary References
... Expression across all tissues indicates that four genes encode cytoplasmic actins (Fig. 3). Protein sequence comparisons placed all B. glabrata actins as most closely related to mammalian cytoplasmic rather than sarcomeric actins (Supplementary Note 30; Supplementary Data 39), a pattern also observed for all six actin genes of D. melanogaster 44 . The actin genes of B. glabrata and other molluscs were most similar to paralogs within their own genomes, rather than to other animal orthologs (Fig. 3). ...
Biomphalaria snails are instrumental in transmission of the human blood fluke Schistosoma mansoni. With the World Health Organization's goal to eliminate schistosomiasis as a global health problem by 2025, there is now renewed emphasis on snail control. Here, we characterize the genome of Biomphalaria glabrata, a lophotrochozoan protostome, and provide timely and important information on snail biology. We describe aspects of phero-perception, stress responses, immune function and regulation of gene expression that support the persistence of B. glabrata in the field and may define this species as a suitable snail host for S. mansoni. We identify several potential targets for developing novel control measures aimed at reducing snail-mediated transmission of schistosomiasis.
... Drosophila melanogaster has six actin isoforms, each of which accumu lates in distinct temporal and spatial patterns. Each isoform has a unique primary sequence, but all are similar to those of vertebrate nonmuscle actins (Fyrberg et al 1981). Gene isolation and characterization revealed that the isoforms are encoded by a closely related family of six genes: Act5C, Act42A, Act57B, Act79B, Act87E, and Act88F, each member of which is named for its chromosomal location (Tobin et al 1980;Fyrberg et al 1980). ...
... They used this background to perform a deficiency screen for modulators of IRS size and number of rhabdomere attachments. They identified Actin5C in their screen, one of the 2 ubiquitously expressed Actin genes (Fyrberg et al. 1981;Fyrberg et al. 1983;Wagner et al. 2002). ...
Rumi is a protein O-glucosyltransferase that adds the sugar O-glucose onto the serine in the target sequence C-S-X-S-(P/A)-C found within properly folded EGF repeats. It was first discovered to modify the Drosophila Notch extracellular domain and to be required for Notch signaling in a temperature dependent manner, but other targets of Rumi remained unknown. Several other proteins in the Drosophila proteome harbor multiple consensus sequence highly predictive of O-glucose, including the transmembrane protein Crumbs and the secreted protein Eyes shut (Eys). Both of these proteins are required for proper eye development and mutations in their human homologs cause a blindness disorder named retinitis pigmentosa. Therefore, we sought to determine whether Rumi plays a role in photoreceptor development. We found that rumi–/– animals have defects in photoreceptor spacing in which many neighboring rhabdomeres are attached. This phenotype cannot be explained by the loss of O-glucose on Notch or Crumbs. However, eys genetically interacts with rumi, and in rumi–/– animals at the start of rhabdomere separation, Eys accumulates intracellularly and decreased levels of Eys reach the extracellular space. Overexpressing a mutant Eys transgene which contains no intact O-glucosylation sites also results in intracellular accumulation of Eys, suggesting that loss of O-glucose from Eys is the cause. Additionally, both the intracellular accumulation and the rhabdomere attachment defect grow more severe at higher temperatures, and Eys degrades at higher temperatures in rumi–/–. In addition, removing one copy of the chaperone Hsc70-3 enhances the rumi–/– phenotype. Together, these data suggest that loss of O-glucose from Eys causes a defect in its proper folding, which leads to decreased Eys reaching the extracellular space and therefore a failure in full separation of the rhabdomeres.
... Fyrberg and collaborators cloned all of them. In a later paper, we noted by DNA sequencing that all of the N-terminal sequences of the six genes were similar to those of vertebrate cytoskeletal actins (14). To my knowledge, it is not known which of the six code for skeletal muscle actins. ...
[Figure: see text]
▪ Abstract Norman Davidson’s training as a physical chemist led him to make key early contributions to the chemistry of DNA. He described the details of DNA denaturation and renaturation, concepts that still form the basis for understanding hybridization. He also applied the single-molecule resolution of the electron microscope to describing the chemistry of circular DNA, mapping specific genes, and characterizing heteroduplexes. The latter became a dominant tool for the study of nucleic acids and contributed to our knowledge of transcription, polyadenylation, and retroviral structure. The advent of cDNA cloning and restriction enzymes enabled Davidson to describe the diversity of Drosophila actin genes and to isolate the gene encoding cAMP phosphodiesterase. Davidson then turned his attention to neuroscience and participated in cDNA cloning, oocyte expression, and structure-function studies of nicotinic acetylcholine receptors, voltage-gated sodium channels, a GABA transporter, a G protein-gated potassium channel, and calcium channels. His interests also extended to synaptic plasticity, and he helped to define the role of neuronal nitric oxide synthase and of trkB receptors. His final experiments concerned the role of protein kinase A in long-term potentiation. (The abstract was written posthumously by a colleague.)
... Comparison of amino-terminal sequence of vertebrate and Drosophila actins with flightin. Vertebrate sequences are from Vanderkerckhove and Weber (56)(57)(58) and Drosophila sequences are from Fyrberg et al. (13). Since the first two amino acids of Drosophila actins (Met and Cys) are removed posttranslationally, the aspartic acid at position 3 is the NH2-terminal amino acid of the mature protein (37). ...
The indirect flight muscles of Drosophila are adapted for rapid oscillatory movements which depend on properties of the contractile apparatus itself. Flight muscles are stretch activated and the frequency of contraction in these muscles is independent of the rate of nerve impulses. Little is known about the molecular basis of these adaptations. We now report a novel protein that is found only in flight muscles and has, therefore, been named flightin. Although we detect only one gene (in polytene region 76D) for flightin, this protein has several isoforms (relative gel mobilities, 27-30 kD; pIs, 4.6-6.0). These isoforms appear to be created by posttranslational modifications. A subset of these isoforms is absent in newly emerged adults but appears when the adult develops the ability to fly. In intact muscles flightin is associated with the A band of the sarcomere, where evidence suggests it interacts with the myosin filaments. Computer database searches do not reveal extensive similarity to any known protein. However, the NH2-terminal 12 residues show similarity to the NH2-terminal sequence of actin, a region that interacts with myosin. These features suggest a role for flightin in the regulation of contraction, possibly by modulating actin-myosin interaction.
... Physarum Tubulins RNA (T . Schedl, unpublished) ; (b) a chick ß-tubulin cDNA clone, pT2, a generous gift of D. Cleveland (9), and, on a separate filter, a Drosophila 0tubulin genomic clone, pDTB4, a generous gift of S. Natzle (35); (c) a l.8-kb fragment containing the entire coding sequence from the actin genomic clone ADmA2, a generous gift of E. Fyrberg (12,13); (d) pBR322 vector . To each filter was hybridized 2 kg of poly A-containing RNA isolated from late G2 phase plasmodia. ...
Three alpha-tubulins and two beta-tubulins have been resolved by two-dimensional gel electrophoresis of whole cell lysates of Physarum myxamoebae or plasmodia. Criteria used to identify the tubulins included migration on two-dimensional gels with myxamoebal tubulins purified by self-assembly into microtubules in vitro, peptide mapping with Staphylococcus V8 protease and with chymotrypsin, immunoprecipitation with a monoclonal antibody specific for beta-tubulin, and, finally, hybrid selection of specific mRNA by cloned tubulin DNA sequences, followed by translation in vitro. Differential expression of the Physarum tubulins was observed. The alpha 1- and beta 1-tubulins were detected in both myxamoebae and plasmodia; alpha 2 and beta 2 were detected only in plasmodia, alpha 3 was detected only in the myxamoebal phase, and may be specific to the flagellate. Observation of more tubulin species in plasmodia than in myxamoebae was remarkable; the only microtubules detected in plasmodia are those of the mitotoic spindle, whereas myxamoebae display cytoplasmic, centriolar, flagellar, and mitotic-spindle microtubules. In vitro translation of myxamoebal and plasmodial RNAs indicated that there are distinct mRNAs, and therefore probably separate genes, for the alpha 1-, alpha 2-, beta 1-, and beta 2-tubulins. Thus, the different patterns of tubulin expression in myxamoebae and plasmodia reflect differential expression of tubulin genes.
... Genes for several muscle proteins of Drosophila have now been cloned. All of the major myofibrillar proteins that have been sequenced, actin, tropomyosin, myosin heavy chain, myosin light chain 2, and myosin light chain 3, show a high level of sequence homology with their vertebrate counterparts (Fyrberg et al., 1981;Bernstein et al., 1983;Basi et al., 1984;Karlik et al., 1984;Falkenthal et al., 1985;Parker et al., 1985;Toffenetti et al., 1987). In contrast, the mp20 protein sequence shows no strong homology with any known protein but has two regions with similarity to a number of known calcium binding sites. ...
A Drosophila melanogaster gene encoding a muscle specific protein was isolated by differential screening with RNA from primary cultures of myotubes. The gene encodes a 20-kD protein, muscle protein 20 (mp20), that is not detected in the asynchronous oscillatory flight muscles, but is found in most, if not all, other muscles (the synchronous muscles). The sequence of the protein, deduced from the DNA, contains two regions of 12 amino acids with significant similarity to high-affinity calcium-binding sites of other proteins. This protein is easily extracted from the contractile apparatus and thus does not seem to be a tightly bound structural component. The gene (located in polytene region 49F 9-13) is unique in the D. melanogaster genome and yields two transcripts, 1.0 and 0.9 kb long. The levels of the two transcripts are regulated differently during development, yet the coding regions of the two transcripts are identical.
... Analysis of overlapping deletions and testing of individual mutants for genes within the deleted interval mapped the responsible locus to Actin5C (Act5C), as alleles of Act5C as well as mild RNAi knockdown of Act5C in the EP-TH background completely phenocopied the phenotype observed with the deficiency ( Figure 1D, E and Figure S1). In Drosophila there are six actin genes: Act5C and Act42A are ubiquitously expressed, while Act57B, Act79B, Act87E, and Act88F are muscle-specific [24][25][26]. To test the specificity of our enhancement, we re-examined deletions that individually remove each actin gene. ...
Multicellular tubes consist of polarized cells wrapped around a central lumen and are essential structures underlying many developmental and physiological functions. In Drosophila compound eyes, each ommatidium forms a luminal matrix, the inter-rhabdomeral space, to shape and separate the key phototransduction organelles, the rhabdomeres, for proper visual perception. In an enhancer screen to define mechanisms of retina lumen formation, we identified Actin5C as a key molecule. Our results demonstrate that the disruption of lumen formation upon the reduction of Actin5C is not linked to any discernible defect in microvillus formation, the rhabdomere terminal web (RTW), or the overall morphogenesis and basal extension of the rhabdomere. Second, the failure of proper lumen formation is not the result of previously identified processes of retinal lumen formation: Prominin localization, expansion of the apical membrane, or secretion of the luminal matrix. Rather, the phenotype observed with Actin5C is phenocopied upon the decrease of the individual components of non-muscle myosin II (MyoII) and its upstream activators. In photoreceptor cells MyoII localizes to the base of the rhabdomeres, overlapping with the actin filaments of the RTW. Consistent with the well-established roll of actomyosin-mediated cellular contraction, reduction of MyoII results in reduced distance between apical membranes as measured by a decrease in lumen diameter. Together, our results indicate the actomyosin machinery coordinates with the localization of apical membrane components and the secretion of an extracellular matrix to overcome apical membrane adhesion to initiate and expand the retinal lumen.
... Comparisons of nucleotide sequences from the protein coding regions and exon-intron arrangements of related genes provide a means of tracing their evolution pathways [17,18]. Before the advent of the era of large-scale sequencing, actin gene family has been investigated in many organisms [19][20][21][22][23][24]. Those results indicate that actin gene family is highly conserved, and the number of actin genes among these organisms is variable. ...
Actin is one of the most highly conserved proteins and plays crucial roles in many vital cellular functions. In most eukaryotes, it is encoded by a multigene family. Although the actin gene family has been studied a lot, few investigators focus on the comparison of actin gene family in relative species. Here, the purpose of our study is to systematically investigate characteristics and evolutionary pattern of actin gene family in primates. We identified 233 actin genes in human, chimpanzee, gorilla, orangutan, gibbon, rhesus monkey, and marmoset genomes. Phylogenetic analysis showed that actin genes in the seven species could be divided into two major types of clades: orthologous group versus complex group. Codon usages and gene expression patterns of actin gene copies were highly consistent among the groups because of basic functions needed by the organisms, but much diverged within species due to functional diversification. Besides, many great potential pseudogenes were found with incomplete open reading frames due to frameshifts or early stop codons. These results implied that actin gene family in primates went through "birth and death" model of evolution process. Under this model, actin genes experienced strong negative selection and increased the functional complexity by reproducing themselves.
... Although the structure of a functional human a-actin gene has not been determined yet this gene is likely to be split since all vertebrate actin genes studied so far, including a human cardiac muscle actin gene and a a-actin gene from chick and rat, are interrupted by several introns (29)(30)(31). In fact, with the exception of the genes in the slime mold Dictyostelium (32) and an actin gene in the fission yeast Schizosaccharomyces pombe (Mertins and Gallwitz, unpublished), the actin genes of all other eukaryotes that have been studied contain intervening sequences (26,(33)(34)(35)(36). It is therefore most likely that the human f-actin pseudogene that we have described here has lost its intervening sequence(s) and represents a so-called processed gene (9). ...
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.
... Using the synthesized cDNA and A. luxuriosa defensin 1-specific primers (forward primer 1DefFullS, reverse primer 1DefFullA), 20, 25, or 30 cycles of RT-PCR were performed at 95°C for 1 min, 40°C for 1 min, and 72°C for 1 min. As an internal marker, insect actin (Fyberg et al., 1981) was amplified with primers 5Ј-AACT-GGGACGACATGGAGAAGATCTGGCA-3Ј (forward primer) and 5Ј-GAGATCCACATCTGCTG-GAAGGTGGACAG-3Ј (reverse primer). The effectiveness of the DNase treatment of total RNA was confirmed by running a PCR with insect actin primers in the absence of reverse transcriptase to ensure that there was no contamination by genomic DNA. ...
We purified an antibacterial peptide from the larval hemolymph of a coleopteran beetle, Acalolepta luxuriosa. Using structure analysis and cDNA cloning, this peptide was identified as a novel member of the insect defensin family and named A. luxuriosa defensin 1. A. luxuriosa defensin 1 shares high sequence similarity with other defensins, especially coleopteran defensins. A dendrogram of coleopteran insect defensins based on sequence homology revealed that A. luxuriosa defensin is closer to Tenebrionoidea defensin than to Scarabaeoidea defensin, which parallels the evolutionary relationship of these coleopteran insects. Although A. luxuriosa defensin 1 was most homologous with Tenebrio molitor tenecin 1, its antibacterial spectrum was broader, affecting the growth of Gram-positive and Gram-negative bacteria, suggesting that the variability of the antibacterial spectrum results from small sequence differences.
... The deduced amino acid sequence of Didactl is 376 amino acids long . It is a typical invertebrate actin (Fyrberg et al., 1981) resembling more the cytoplasmic than the muscular isoforms of vertebrate actins . Didactl is for example 96% homologous to human cytoplasmic -y-actin but only 92 .6% ...
We have examined actin cDNA of the flatworm Diphyllobothrium dendriticum (Cestoda). Actin is a contractile protein that has been implicated in a variety of developmental and cellular processes. It is highly conserved and present in all eukaryotic cells. It is of particular interest to analyze evolutionary preserved genes in flatworms, because ancestral flatworms are regarded to play a central role in the evolution of the metazoans (Barnes et al., 1998). Screening a cDNA library of D. dendriticum (UniZap XR, Stratagene) with a human ?-actin probe resulted in several positive clones. One of the cDNA inserts, Didactl, consisting of 1392 bp was completely sequenced. The established nucleotide sequence revealed a 5' untranslated region of 33 bp, the entire open reading frame of 1128 bp and a 3' untranslated region of 231 bp which ends in a stretch of 21 A residues. The potential polyadenylation signal (AATAAA) is located 14 bp upstream of the poly (A) tail. The deduced amino acid sequence of Didactl is 376 amino acids long. It is a typical invertebrate actin (Fyrberg et al., 1981) resembling more the cytoplasmic than the muscular isoforms of vertebrate actins. Didactl is for example 96% homologous to human cytoplasmic ?-actin but only 92.6% identical with human smooth muscle d-actin. The actin proteins are generally encoded by a multigene family which differs in size from species to species. Most organisms have four to eight genes coding for actin in their genome, but the number of actin genes can also be over 20 (Hamelin et al., 1988). Sequence comparisons of Didactl and the partly sequenced cDNA clones indicate that D. dendriticum has at least four different genes coding for actin in its genome.
... The 3' noncoding nucleotides in the a and a'-subunit cDNAs show more sequence conservation in the 3' noncoding region than the adjacent coding sequences, which suggests that some functional constraint has prevented the divergence of these sequences. The genes for the globins of distantly related organisms (23), the actins of Drosophila and sea urchin (30,31) and other groups of closely related proteins vary significantly more in their 3' noncoding sequences than do the noncoding sequences of plant genes. In contrast, the globin genes of closely related primates and some immunoglobin genes of mice exhibit a similar degree of nucleotide conservation in their 3' noncoding region (32,33). ...
Nineteen cloned cDNAs encoding the α and α‘ –subunits of the 7S seed storage protein in the soybean, Glycine max, have been isolated from a recombinant cDNA library constructed with mRNA from maturing seeds. In addition, a gene encoding
an α‘ –subunit has been isolated from a recombinant Charon 4A phage library containing genomic Glycine max DNA. The cloned DNAs have been divided, on the basis of their endonuclease sites, into two main classes of sequences which
differ in approximately 6% of their nucelotides. Whereas the proteins encoded within each DNA class are nearly identical,
the proteins encoded by the two different classes of soybean DNAs are distinct and correspond to α and α‘ –subunits. Thus,
the α and α‘ ’ subunits are coded for by two closely related multigene families. The amino acid differences in the portions
of the α and α’ –subunits presented in this paper occur primarily near the carboxyl-terminus. The 3‘ noncoding nucleotides
of the cloned α and α’ –subunit DNAs are more highly conserved than are the coding nucleotides. This conservation suggests
that the 3’ untranslated sequences of the α and α’ –subunit mRNAs are functional in the expression of the α and α’ –subunit
proteins or in the stabilization of the 7S subunit mRNAs.
... Clones of A gambiae actin genes were obtained by PCR using a pair of primers designed around two evolutionary conserved actin gene regions that are rich in amino acids encoded by only one or two codons. The regions selected represent amino acids 81-88 (WDDMEKIWH) and amino acids 405-414 (MYPGIADRMQ) in published Drosophila melanogaster and Bombyx mori actin sequences (Fyrberg et al., 1981;Mounier & Prudhomme, 1986). The two oligonucleotide primers included deoxyinosone residues (I) at all ambiguous positions and extra nucleotides at the S' ends that provided restriction sites and CGC clamps for cloning of the products. ...
Five actin genes have been identified in the mosquito Anopheles gambiae, and a constitutively expressed actin gene has been chosen for detailed analysis. We have physically mapped and sequenced this gene and six associated cDNAs, including translated coding regions, as well as the 5 and 3 flanking sequences. Analysis of stage-specific RNA shows this gene to be present in all stages of mosquito development and in an established A. gambiae cell line, thus indicating a cytoskeietal actin. In the sequence of the translated coding region and in pattern of expression, this gene is very similar to the cytoskeietal actin genes of Droso-phila melanogaster, and in sequence, equally similar to the Artemia cytoskeietal actin gene 403 (99.2% identity among the three amino acid sequences). Sequencing of this A. gambiae actin gene (designated actWior its location in chromosome division 1D) and selected cDNAs shows that it possesses three alternative leader sequences; thus the gene appears to have three alternative promoters. These promoters should ultimately prove useful in the production of transgenic constructs for constitutive expression.
We describe a method for examining the in vivo distribution of a protein on specific eucaryotic DNA sequences. In this method, proteins are cross-linked to DNA in intact cells, and the protein-DNA adducts are isolated by immunoprecipitation with antiserum against the protein. Characterization of the DNA cross-linked to the precipitated protein identifies the sequences with which the protein is associated in vivo. Here, we applied these methods to detect RNA polymerase II-DNA interactions in heat-shocked and untreated Drosophila melanogaster Schneider line 2 cells. The level of RNA polymerase II associated with several heat shock genes increased dramatically in response to heat shock, whereas the level associated with the copia genes decreased, indicating that both induction of heat shock gene expression and repression of the copia gene expression by heat shock occur at the transcriptional level. Low levels of RNA polymerase II were present on DNA outside of the transcription units, and for at least two genes, hsp83 and hsp26, RNA polymerase II initiated binding near the transcription start site. Moreover, for hsp70, the density of RNA polymerase II on sequences downstream of the polyadenylate addition site was much lower than that observed on the gene internal sequences. Examination of the amount of specific restriction fragments cross-linked to RNA polymerase II provides a means of detecting RNA polymerase II on individual members of multigene families. This analysis shows that RNA polymerase II is associated with only one of the two cytoplasmic actin genes.
Transcription from reticuloenodotheliosis virus strain T (REV-T), an avian retrovirus unrelated to avian leukosis and sarcoma viruses, is modulated by sequences in at least five functional domains. A promoter containing a TATA and multiple CCAAT motifs in U3 of the long terminal repeat was absolutely required for transcription. Transcriptional efficiency was greatly augmented by an enhancer immediately upstream, which contained a 22-base-pair repeated sequence. Transcription was further influenced by a negative-acting domain in the 5' region of U3 and two downstream domains in the transcribed non-protein-coding region. One of these latter domains contained a consensus enhancer core sequence and positively affected transcription in both mammalian and avian cells; the other acted negatively in a dog cell line. Transcription from REV-T in vivo required cellular factors which could be competed for specifically by the promoter or enhancer domain. The downstream domains competed with reporter genes containing these domains, but not directly with the U3 sequences. The promoter, enhancer, and the positive-acting downstream domains formed multiple complexes with distinct classes of cellular factors in both avian and mammalian cell extracts. Binding of factors to the promoter and enhancer domains was cooperative when these domains were joined in cis.
We have purified a novel antibacterial peptide from the hemolymph of the coleopteran insect Acalolepta luxuriosa, of the family Cerambyocidae, and named it luxuriosin. This peptide showed growth-inhibitory activity against Micrococcus luteus and germination- and/or growth-inhibitory activity against the conidia from rice blast fungus, Magnaporthe grisea. The amino acid sequence determined by cDNA cloning identified luxuriosin as a peptide of 88 amino acids with a theoretical molecular weight of 10,368.34, containing a Kunitz domain.
In considering the mechanisms which could have led to the amazing variety of biological structures in the animal world, it has seemed reasonable to focus on the evolution of genomic regulatory pathways ((50), and Britten, this volume). Biological structure is the immediate result of developmental processes in which sets of diverse structural genes are expressed as ontogenically functional units. Such units must include genes that control the cell divisions in given tissue anlage or cell lineages, the nature of the products formed in the cells, their interactions with adjacent cells, and so forth. Relatives of most known structural genes seem to occur in a very wide phylogenetic range of creatures, while the specific patterns of development in which they are utilized are characteristic of each taxonomic group of organisms. If we understood the genomic organization underlying these specific ontogenic regulatory patterns, we might be in an excellent position to construct a useful theory of evolutionary invention at the DNA level. Unfortunately, such understanding still lies beyond current knowledge, though perhaps not very far beyond. In this essay I have chosen somewhat arbitrarily to consider two forms of hypothetical genomic regulatory organization. For neither of these is there yet any very strongly convincing evidence, particularly in regard to ontogenic regulatory coordination. Nonetheless, it is interesting to examine the implications for both kinds of proposed regulatory organization of various kinds of genomic alteration known to occur in evolution at a relatively high rate. This heuristic exercise shows that it is not difficult to imagine evolutionary mechanisms that could have had large scale functional effects on ontogenic patterns, and thus have led to the appearance of novel biological structures.
Actin is a ubiquitous and highly conserved protein found in eukaryotic organisms. In this study, we describe the cDNA cloning and mRNA expression of an actin gene from the mulberry longicorn beetle, Apriona germari. The A. germari actin cDNA is 1524 bp containing a complete 1128 bp open reading frame that encodes a polypeptide of 376 amino acid residues with a predicted molecular weight of about 41.5 kDa. The deduced amino acid sequence of the A.germari actin cDNA showed 99% protein sequence identity to Homalodisca coagulata actin, differing at only two amino acid positions, and 92-98% protein sequence identity to known insect species actins. The predicted three-dimensional structure of A. germari actin revealed the four residue hydrophobic pulg loop characteristic of the actin family. Northern blot analysis showed that A. germari actin is highly expressed in epidermis and muscle, and less strongly in midgut, but not in the fat body of A. germari larva.
Objective
To obtain the complete β-actin gene from Aedes albopictus.
Summary Evolutionary studies on the tubulin multigene families were initiated by nucleotide sequence analysis of cDNA clones complementary to sea urchin (Lytechinus pictus) tesits α- and β-tubulin mRNAS. Sequence comparisons of three partial β-tubulin cDNA clones (pβ1, pβ2, pβ3) demonstrated the existence of tubulin mRNA heterogeneity. pβ2 and pβ3 contain identical tubulin-coding regions and extremely similar 3′ untranslated sequences, including a polyadenylation signal (AAUAAA). However, pβ2 contains an additional region of 3′ untranslated sequence which includes a second plyadenylation signal. These two sequences may be allelic, representing products of alternative transcription termination or processing pathways. pβ1 and pβ2 (or pβ3) cDNAs almost certainly correspond to transcripts of distinct but evolutionarily related genes. Examination of the available coding portions showed that they differ only by a few silent nucleotide substitutions and the deletion/insertion of one codon; most of the differences are clustered within the last 15 3′-end codons. In contrst, their 3′ untranslated sequences are considerably divergent. Nucleotide alignment in this region was feasible by considering specific point and segmental mutations, mainly T↔C transitions and small deletions/insetions associated with small direct repeats. The sea urchin α- and β-tubulin cDNA and corresponding protein sequences were compared with previously described tubulin cDNA and protein sequences from other organisms. Both α and β tubulins are very conserved proteins, evolving with a rete comparable to that of histones. Analysis of the nucleotide divergence of the coding cDNA regions showed that relacement sites have changed with a rate 20–175 times lower than that of the silent sites. Among the 177 codons compared between the sea urchinb testic and chick brain β-tubulin cDNAs, there are 7 conservative amino acid replacements and the deletion/insertion of two codons. Most of these changes are clustered near the C-terminus. The 161-amino acid portion of chick brain, rat and porcine α-tubulin sequences differs by 3 conservative amino acid replacements from the corresponding sea urchin testis α-tubulin sequence. The compared interspecies 3′ untranslated sequences are very divergent.
To obtain the complete β-actin gene from Aedes albopictus.
We have utilized the rabbit 6-day blastocyst as a model system in which to examine the effect of environmental stress on embryonic gene expression. Elevation of the incubation temperature from 37 to 43 °C, exposure to 50 μM sodium arsenite or mechanical injury (resulting in the structural collapse of the 6–day rabbit blastocyst) was found to depress total protein synthesis as well as enhance the synthesis of a 70,000-dalton stress-induced protein. The molecular mass of this stress protein is similar to a heat shock protein (HSP) found in other eukaryotic systems. A recombinant DNA probe consisting of the 5′ end of a mouse gene for a 70,000-dalton HSP hybridized to RNA isolated from heat shocked, sodium arsenite-treated, and mechanically injured blastocysts but not to RNA isolated from control embryos. These results as well as in vitro translation data suggest that the expression of the 70 K HSP is controlled at the transcriptional level. The levels of actin mRNA, as detected by means of a recombinant DNA probe encoding a Drosophila actin gene, did not undergo a major alteration following these different stresses. The relevance of these observations to embryonic cellular homeostatis is discussed.
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 nucleotide sequence of the rat β-actin gene was determined. The gene codes for a protein identical to the bovine β–actin.
It has a large intron in the 5’ untranslated region 6 nucleotides upstream from the initiator ATG, and 4 Introns 1n the coding
region at codons specifying amino acids 41/42, 121/122, 267, and 327/328. Unlike the skeletal muscle actin gene and many other
actin genes, the 6-act1n gene lacks the codon for Cys between the Initiator ATG and the codon for the N-terminal amino acid
of the mature protein.
The usage of synonymous codons 1n the β–actin gene is nonrandom, and is similar to that in the rat skeletal muscle and other
vertebrate actin genes, but differs from the codon usage in yeast and soybean actin genes.
Genomic andcDNAsequencing studies showthattranscripts fromthemuscle myosin heavy-chain (MHC) geneofDrosophila melanogaster arealternatively spliced, producing RNAsthatencode atleast twoMHC isoforms withdifferent C termini. Transcripts encoding anMHC isoform with27unique C-terminal amino acids accumulate during bothlarval andadult muscle differentiation. Transcripts forthesecondisoform encodeoneunique C-terminal aminoacidandaccumulate almost exclusively inpupalandadult thoracic segments, thelocation oftheindirect flight muscles. The3'splice acceptor site preceding thethorax-specific exonisunusually purine richandthusmayserve asathorax-specific splicing signal. We suggest thatthe alternative C termini ofthese twoMHC isoforms control myofilament assembly andmayplayarolein
Drosophila cell lines respond to physiological doses of 20-OH-ecdysone by entering mitotic arrest and differentiating morphologically. The cells also exhibit changes in gene expression. Several enzyme activities are induced, and the synthesis of cytoplasmic actin and of the four small heat-shock proteins (hsp) is initiated. Hybrid genes, containing the 5′ region of Drosophila heat-shock protein genes ligated to the herpes simplex virus thymidine kinase gene (tk), have been transfected into cells of the Drosophila cell line S3. Constructions containing sequences upstream from hsp 70, or from any of the small hsp genes, show heat-inducible tk expression. Ecdysterone-inducible tk expression is seen only in transfections with small hsp-tk hybrid genes. This transient expression system can be used as an assay for function to define regions of DNA, flanking the coding region of inducible genes, which are necessary for normal gene expression and gene regulation in cultured cells.
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.
▪ Abstract The generation of isoforms via gene duplication and alternative splicing has been a valuable evolutionary tool for the creation of biological diversity. In addition to the formation of molecules with related but different functional characteristics, it is now apparent that isoforms can be segregated into different intracellular sites within the same cell. Sorting has been observed in a wide range of genes, including those encoding structural molecules, receptors, channels, enzymes, and signaling molecules. This results in the creation of intracellular compartments that (a) can be independently controlled and (b) have different functional properties. The sorting mechanisms are likely to operate at the level of both proteins and mRNAs. Isoform sorting may be an important consequence of the evolution of isoforms and is likely to have contributed to the diversity of functional properties within groups of isoforms.
A clone of the Drosophila melanogaster (Meigen) gene Act5C was used to isolate an actin gene from a Hessian fly, Mayetiola destructor (Say), genomic library in phage lambda. A combined molecular and cytological analysis with the Hessian fly actin gene (designated MdA1) was undertaken. The coding region of this gene was contiguous and encoded a protein that was 99% identical to the intersegmental muscle actins 57A and 87E from D. melanogaster. Additionally, the protein was >97% identical to the flight-muscle-specific actin 88E from D. melanogaster as well as muscle actins 1 and 2 from Bombyx mori (L.). Only the muscle actin 79B from D. melanogaster, a muscle actin in leg and thorax, showed <97% identity with actin 1 from Hessian fly. The actin 1 from Hessian fly shared less amino acid identity (94–95%) with the cytoplasmic actins from D. melanogaster, Anopheles gambiae (Giles), and B. mori, with differences occurring in a conserved region of the cytoplasmic actins proposed to function in interaction with actin binding proteins. These results are consistent with the Hessian fly MdA1 gene encoding a muscle actin. When compared with the D. melanogaster Act57A and Act87E genes, the Hessian fly MdA1 gene revealed 81% identity at the nucleotide level, with most of the variation associated with third-codon-position G+C content. The MdA1 gene also had a high degree of general synonymous codon usage bias as measured by scaled chi-square. Southern gel blot analyses as well as in situ hybridization on salivary polytene chromosomes with MdA1 as the probe revealed that a gene family with at least five members encodes the actins in Hessian fly. Future identification of promoters for actins from Hessian fly should prove useful for gene expression in transgenic constructs.
An actin-encoding cDNA of the kuruma shrimp (Marsupenaeus japonicus) was cloned from a hemocyte cDNA library. To obtain the full length of actin cDNA, a partial cDNA fragment of 517 bp was isolated by reverse transcriptase-polymerase chain reaction (RT-PCR) from poly (A)+ RNA derived from hemocytes and used as a probe for plaque hybridization. The full actin cDNA was 1,327 bp in length, encoding 376 amino acid residues. The deduced amino acid sequence had 100% identity to that of β-actin from the Pacific white shrimp Litopenaeus vannamei, and extremely high similarity to the sequences of actins in other vertebrates and invertebrates. The cDNA fragments of the actin were amplified by RT-PCR from all organs tested: lymphoid organ, stomach, hepatopancreas, heart, kidney, muscle, eye, ovary, and testis.
Tissue growth in Crustacea occurs at specific stages of the moult cycle and is influenced by a number of physical, hormonal and environmental factors. In order to understand the mechanisms responsible for controlling intermittent muscle growth in Crustacea, the effects of various factors on rates of protein synthesis and gene expression for the myofibrillar proteins, have been examined. These studies include the effects of mechanical stretch on muscle fibres; the influence of the moulting hormones, ecdysteroids; and the effect of temperature which is an important environmental variable. Sarcomeric proteins have been cloned and used to measure mRNA levels of actin, myosin HC and tropomyosin in various muscles over the moult cycle. Results from these studies demonstrate that both transcriptional and translational regulation occurs in response to hormonal and mechanical stimulation. Temperature has a direct effect on rates of protein synthesis and transcription in intermoult muscles but overall protein turnover may remain unchanged due to a concomitant increase in protein degradation rates.
The deposition and apolysis of insect cuticles have long been known to be regulated by ecdysone. Unsclerotized, chitin-containing procuticles contain evolutionarily conserved, hydrophobic proteins that are soluble in solutions of denaturing agents. The pupal procuticle of Drosophila is deposited by larval and imaginal epidermis starting 9 h after puparium formation when the ecdysone titer is low. Initially, a set of low molecular weight proteins (less than 25,000 daltons; low molecular weight pupal cuticle proteins = S-PCPs) is synthesized. However, about the time of pupation, synthesis of S-PCPs ceases, and high molecular weight proteins (greater than 50,000 daltons; H-PCPs) are synthesized. In vitro experiments indicate that the initial formation of the procuticle with synthesis of the S-PCPs requires a pulse of hormone followed by withdrawal (6 h with 20-hydroxyecdysone, 1 μg/ml). The switch from synthesis of S-PCPs to H-PCPs is facilitated by a second, short pulse of 20-hydroxyecdysone (0.1 μg/ml, 3 h). Ultrastructural localization demonstrates that the S-PCPs are located only in the external lamellae of the procuticle, while the H-PCPs are present only in internal lamellae. Developmental analyses with cloned genes indicate that cuticle protein genes are expressed during only one stage of Drosophila development. Some of the genes encoding S-PCPs are limited in their expression to larval (posterior) or imaginal (anterior) epidermis. Preliminary molecular analyses of the larval and pupal cuticle protein genes indicate that they are organized in different ways. For example, four larval genes exist in a cluster with divergent transcription, and one PCP gene, PCP-GART, is located within an intron of a “housekeeping” gene.
1. Two-dimensional electrophoresis was carried out to determine the isoelectric points of actins from thoracic and leg muscles of various kinds of insects (Coleoptera, Orthoptera, Odonata, Hemiptera, Lepidoptera, Hymenoptera and Diptera).2. The isoelectric points of insect muscle actin ranged from 5.6 to 5.9, appreciably more basic than rabbit skeletal muscle actin (α-actin).3. There was usually one predominant isoform of insect muscle actin except that two isoforms were detected in fly and wasp thoracic muscles.4. In a cicada, the isoelectric points of actins from thoracic, leg and sound organ were 5.65, 5.68 and 5.57, respectively.5. Actins from spider thoracic muscle, and crab claw and crayfish claw muscles also showed the isoelectric points of 5.5–5.7.6. Thus arthropod muscle actins appear to have more basic isoelectric points than vertebrate skeletal muscle ones.
The complete 3 untranslated region (3UTR) sequence of the human skeletal-actin gene has been compared with the corresponding regions of the rat and chicken skeletal-actin genes. This comparison reveals that the skeletal-actin 3UTR is composed of conserved and nonconserved segments. By using genomic Southern transfer blots and thermal stability (Tm) measurements, we found that the cardiac-actin gene 3UTR also consists of conserved and nonconserved segments. Comparison of human andXenopus laevis cardiac-actin mRNA sequences confirms the presence of a region of high similarity in the 3UTR. We conclude that subsegments of the 3UTRs of both skeletal- and cardiac-actin genes of birds and mammals are under considerable selective pressure. This suggests that these conserved sequences may have functional roles in actin-gene expression or regulation, and that these roles might be different for each actin isoform.
Smooth muscle actin (Actg) is expressed in smooth muscle and in haploid male germ cells. In order to further characterize theActg gene, a 60-nucleotide-long isotype-specific probe was synthesized. Single bands of DNA were detected when this oligonucleotide was used to probe blots of mouse genomic DNA digested with PstI, EcoRI, KpnI, or XbaI. These results suggestActg is a single-copy gene with no detectable pseudogenes. TheActg gene was mapped to mouse chromosome 6 by Southern blot analysis of DNA isolated from 15 mouse-hamster hybrid cell lines.
The tissue and developmental specificities of the three Drosophila isoactins, originally identified in primary myogenic cultures and in the permanent Schneider L-2 cell line, have been investigated. Of these three isoactins (I, II, and III), actins I and II are stable and actin III is unstable. Two-dimensional polyacrylamide gel electrophoretic analyses of total cellular extracts after 1-h [(35)S]methionine pulses were performed on a large variety of embryonic, larval, and adult muscle and nonmuscle tissues. The results suggest that isoactins II and III are generalized cellular actins found in all drosophila cell types. Actin I, on the other hand, is muscle-associated and is found exclusively in supercontractile muscle (such as larval body wall and larval and adult viscera) including primary myogenic cell cultures. Although actin I synthesis is not detectable during very early embryogenesis, it is detectable by 25 h and actin I is a major stable actin in all larval muscle tissues. Actin I is synthesized in reduced amounts relative to the other actins in late third instar larvae but is again a major product of actin synthesis in the adult abdomen. A stable actin species with the same pI as actin III has been identified in the adult thorax and appears to be unique to flight muscle tissue. This new stable form of thoracic actin may be the result of a stabilization of the actin III found in other tissues or may be an entirely separate gene product.
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The observation of Thomas et al. (1970) that certain double-stranded DNA fragments from eukaryotic chromosomes can form circular structures after treatment with exonucleases (Fig. 1, first two steps) provides a method of considerable potential for the topographical analysis of repetitive sequences. The formation of such “Thomas circles” is expected for fragments derived from either of two topographical classes of repetitive sequences—the tandem repetition and the intermittent repetition. In the tandem repetition, a given sequence is serially repeated without contamination with other sequences. This appears to be the case for the small, highly repetitive sequences in satellite DNAs (Peacock et al., this volume; Gall, 1973; Gall et al., this volume), and for the repeated gene-spacer sequences found in DNAs specifying the 5S and 18–28 S rRNAs (Brown and Sugimoto, this volume). In the intermittent repetition, the repeated sequence is interspersed among sequences that are not repeated, at least within the DNA...
Four distinct actin genes of the sea urchin Strongylocentrotus purpuratus have been isolated from a recombinant Charon 4 phage library of genomic DNA. The four genes differ considerably from each
other in many of their restriction sites. Two of the four genes are closely linked; they are present in the same fragment
of cloned DNA. This fragment has been extensively mapped, and some parts of the DNA have been sequenced. The two linked genes
are oriented in the same direction, separated by 7.5 kb of DNA. One has an intron following the CAG that codes for the glutamine
residue at position 121 in the amino acid sequence of actin. This represents the fifth distinct site at which introns have
been found in actin genes, suggesting that the primordial actin gene had at least 6 exons and 5 introns. The actin genes form
a distinctive family in which most introns have apparently been precisely excised from the genes.