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Molecular structure of the human cytoplasmic ??-Actin gene: Interspecies homology of sequences in the introns
Abstract
A recombinant phage that carries the cytoplasmic beta-actin gene was isolated from a human DNA library. The nucleotide sequence of this gene was determined. The amino acid sequence deduced from the nucleotide sequence matches perfectly that of beta-actin from human fibroblasts. The gene contains five introns. A large intron was found in the 5' untranslated region six nucleotides upstream from the ATG initiation codon. Four introns were found within the coding region at codons specifying amino acids 41/42, 121/122, 267, and 327/328. In contrast to the human cardiac muscle actin gene, the aorta-type smooth muscle actin gene, and the stomach-type smooth muscle actin gene, the beta-actin gene lacks the codon for cysteine between the ATG initiation codon and the codon for the NH2-terminal amino acid of the mature protein. Hybridization of genomic DNA with DNA fragments derived from intron I in the 5' untranslated region and from intron III strongly suggests the presence of a single beta-actin gene in the human genome. The DNA sequences of the coding region, of the 3' untranslated region, and of the sequence block between the "CCAAT" box and "TATA" box in the 5' flanking DNA of the human beta-actin gene are highly homologous to the corresponding sequences of the rat and chicken beta-actin genes. Unexpectedly, the sequence of intron III of the human beta-actin gene shows considerable homology to that of the rat beta-actin gene.
... After washing with DMEM to remove remaining red blood cells, the GC were detached from the flasks with trypsin/ 3 min for the final extension and held at 4°C. The specific sequences for the primers were derived based on the published TRα-1, TRβ-1, TRβ-2, and c-erbAα-2 sequences (Genbank #X74497; Weinberger et al., 1986;Nakai et al., 1988a,b) bp product, whereas amplification of β-actin genomic DNA would yield a 387 bp product (Nakajima-Iijima et al., 1985). Hence, these PCR oligonucleotides also provide an internal control for genomic sulphate (SDS), and 0.1 mg/ml salmon spermatozoa] at 65°C for 1 h, DNA contamination. ...
... using Oligo Primer Analysis Software (National Biosciences Inc, Plymouth, MN, USA)(Table IA). One set of human β-actin primers was used as aIijima et al., 1985). PCR amplification of β-actin cDNA yields a 293 ...
... The ACTB was among the more stable reference genes under all the conditions in this study (Tables 3-5). One of the reasons for this is that ACTIN is the most abundant protein in eukaryotic cells (Nakajima-Iijima et al., 1985). There are at least six ACTIN isoforms in vertebrates: four types in muscle (skeletal, cardiac, vascular smooth, and stomach smooth actins) and two non-muscular types (β-cytoplasmic and γ-cytoplasmic actins) (Vandekerckhove and Weber, 1979), and they are expressed in all types of tissues, as shown in the results of this study, making this gene an optimal normalizer (Tables 3-5). ...
ABSTRACT The objective of the present study was to evaluate the stability of candidate reference genes and select the genes that can be used for normalizing real-time polymerase chain reaction (PCR) in the liver, skeletal muscle, and jejunum tissues of Nellore or Nellore × Angus steers fed different diets. Fourteen purebred and 14 crossbred steers were used, in which half of the animals of each genetic group received a diet containing whole shelled corn (WSC) and the other half whole shelled corn and sugarcane bagasse (WSCB). Stability was analyzed by the RefFinder program. To validate the selection of candidate reference genes, the expression of target genes was evaluated using the different groups of reference genes. The most stable genes were 18S, ACTB, and CASC3 for skeletal muscle; HMBS, ACTB, and 18S for the liver; and GAPDH, ACTB, and CASC3 for the jejunum, regardless of breed and diet provided. Possible errors caused in data analyses were clarified comparing the more and less stable genes as reference for normalization of the target genes FASN, ACOX, SCD1, MGAM, and SLC2A1. The use of the more stable and less stable sets of reference genes may lead to different conclusions in respect to the expression profile of the target studied gene. The selection of more suitable reference genes for each experiment is of utmost importance to ensure the reliability of gene expression studies so that they can be applied in practice.
... We also wanted to investigate transcriptional regulation using LPS as this was the stimulant used in the whole blood culture method. We therefore used (Nakajima-Iijima, 1985). Note the fragment crosses an intron allowing genomic DNA to be identified as a contaminant. ...
This thesis investigates the function of the IL-10 5' flanking region haplotypes and the association of these haplotypes in children with Juvenile Idiopathic Arthritis (JIA). We have shown that children with extended oligoarticular JIA are less likely to have a genotype containing the ATA haplotype than children with oligoarticular JIA (p<0.05, OR = 1.9,95% Cl: 1 to 3.5). The ATA haplotype is associated with lower reporter gene expression in transfection studies (Crawley, 1999c). The ATA/ATA genotype was shown to be associated with lower IL-10 production using whole blood culture in controls (p<0.02). This is consistent with the observation that mean IL-10 production was lower in the parents of those with extended oligoarticular disease when compared to the parents of children with oligoarticular disease (p=0.03, 95% Cl 88 to 2016). A similar effect was seen in asthma where those with severe asthma were less likely to have ATA containing genotypes than controls (p=0.04, OR1.57, Cl: 1.01 to 2). We did not show a difference in genotype distribution between patients with SLE and controls. The transmission disequilibrium test (TDT) demonstrated an increase in transmission of the ATA haplotype to patients with oligoarticular onset JIA (p=0.05). There was also increased transmission of both the ACC and the ATA haplotype to patients with uveitis (p=0.014 for each transmission). Strong linkage disequilibrium was demonstrated (p<0.0005) between these haplotypes and a microsatellite at -1000 in the IL-10 5' flanking region which has previously been shown to be associated with rheumatoid arthritis and systemic lupus erythematosus. Treatment of patients with methylprednisolone was associated with large changes in ex vivo stimulated IL-10 production which was strongly correlated with the change in ESR (correlation coefficient 0.922, p<0.01). To investigate whether the IL-10 5' flanking region haplotypes affected transcription, THP-1 cells were transfected with IL-10 luciferase DNA reporter constructs. Overall, reporter gene expression was higher with the GCC than the ATA construct when the THP-1 cells were transfected using Effectene Liposomes (median fold difference 1.71, range 1.12 to 5.49).
... A semi-quantitative adaptation of the MnSOD RT-PCR was used to estimate the amount of MnSOD transcripts expressed in the samples. The method is based on usage of a housekeeping gene, the gene for -actin (Nakajima-Iijima et al., 1985), as an external standard. The protocol in the -actin RT-PCR was the same as that described for MnSOD, except that the number of PCR cycles was reduced to 20. ...
Proliferation and apoptotic indices of tumour cells may have important prognostic significance. Manganese superoxide dismutase (MnSOD), an important anti‐oxidant enzyme, has been shown to decrease proliferation of malignant cells transfected with the MnSOD gene. The aim of the present study was to investigate the indices of cell proliferation and apoptosis and their prognostic significance in human mesothelioma and to assess the effect of MnSOD on the proliferation and apoptosis of the mesothelioma cells expressing high constitutive MnSOD activity. Tissue sections from 35 subjects with malignant pleural mesothelioma were studied for cell proliferation by Ki‐67 immunohistochemistry and for apoptosis by the TUNEL assay. In additional experiments, 2 mesothelioma cell lines expressing either low (M14K) or high (M38K) MnSOD levels were assessed for proliferative and apoptotic responses to epirubicin. The median proliferation and apoptotic indices of the mesothelioma tissue were 8.2% and 0.75%, respectively. Patients with a high proliferation (>8%) or apoptotic index (>0.75%) showed a worse prognosis (p < 0.001). MnSOD expression was inversely correlated with cell proliferation (p = 0.02). Our cell line experiments indicated that cells expressing high MnSOD levels were more resistant to apoptosis and showed lower proliferation when exposed to epirubicin in vitro. These findings show that high proliferation and apoptosis are associated with a poor prognosis of mesothelioma and that a high MnSOD level is associated with low proliferation of tumour cells. Furthermore, experiments with cultured mesothelioma cells suggest the importance of MnSOD in the proliferation and apoptosis caused by drug exposure. Int. J. Cancer 88:37–43, 2000. © 2000 Wiley‐Liss, Inc.
... Firstly, in order to track the extraction efficiency and to rule out inhibition of the PCR reaction, the presence of DNA was investigated by analysis of the presence of a cytoplasmic human multicopy ß-actin-gene (Nakajima-Iijima et al. 1985). Negative PCR controls were always included. ...
In the Danube Gorges that lie between Serbia and Romania, several archeological sites critical for the understanding of the transitions between the Mesolithic and Neolithic in southeastern Europe have been discovered. In particular, several preserved burial sites, containing around 500 individual skeletal remains, offer a unique opportunity to examine the life-and deathways of these communities. Through an analysis of skeletal remains and patterns of interment, this paper discusses questions of local versus non-local identities, as well as changes in diet throughout the Neolithization. One site in particular, Lepenski Vir, is the basis for research into the paleopathology of local populations. This study concludes that skeletal health parameters suggest a relatively good health status of this population over time, although treponemal infection (a group of diseases including syphilis, bejel, pinta, and yaws) affected large …
... Prior to the multiplex nested PCR, DNA samples amplified the human b-actin gene 30 to ensure the inexistence of amplification inhibitors. ...
Context.—:
Infections are the leading cause of perinatal and infant mortality in low-income and low-resource countries, which have a higher prevalence of infections. Definitive diagnosis of congenital and perinatal infections is largely dependent upon the results of laboratory tests.
Objective.—:
To develop a multiplex nested polymerase chain reaction (PCR) for the simultaneous detection of 7 pathogens containing DNA in their genomes in suspected cases of congenital infection.
Design.—:
Eligible participants were pregnant women with positive immunoglobulin M antibodies raised to one of the pathogens in the prenatal serologic screening, associated or not with fetal ultrasound abnormalities or positive fetal serology. Neonates whose mothers did not attend prenatal care were included when they presented with symptomatology and laboratory parameters suggestive of infection. The detection rate of the multiplex nested PCR was compared with maternal, fetal, and neonatal serology, as well as placental immunohistochemistry and noncommercial amplifications.
Results.—:
Of 161 suspected cases, the multiplex nested PCR detected 60 (37.3%), whereas the tests available in hospital laboratories detected 13 of 60 (21.7%) of the cases detected by the multiplex nested PCR, demonstrating a 4.6 times higher detection rate for the multiplex nested PCR (Fisher exact test, P < .001). Positive amplifications were to Toxoplasma gondii (32 cases), cytomegalovirus (14 cases), parvovirus B19 (5 cases), and adenovirus (5 cases). In 4 cases, 2 pathogens were simultaneously detected. All types of biological matrices were suitable for amplification. Sequencing of multiplex nested PCR products confirmed the molecular findings.
Conclusions.—:
The multiplex nested PCR significantly increased the number of diagnosed congenital infections. Given the scarcity of DNA recovered from amniotic fluid and some neonatal samples, this multiplex nested PCR allows the simultaneous detection of 7 pathogens associated with congenital infections in a reliable, faster, cost-effective, and more sensitive way.
... The list of primers is as follows: rabbit and rat TR4 (6), 5Ј-TTTGTG-GCAGACAAAGATGGA-3Ј and 5Ј-GCCTTGGAATCCGTGGCCA-3Ј; human TR4 (6), 5Ј-TTTGTGGCAGACAAAGATGGA-3Ј and 5Ј-AGC-CTTAGAATCCGTGGCCA-3Ј; mouse TR4 (13), 5Ј-TTTGTGGCAGA-CAAAGATGGA-3Ј and 5Ј-AGCCTTGGAATCCGCGGCCA-3Ј; rabbit and human -actin (14,15), 5Ј-TGGAGAAGAGCTACGAGCTG-3Ј and 5Ј-ACTCGTCATACTCCTGCTTG-3Ј; rat -actin (16), 5Ј-TGGAGAA-GAGCTATGAGCTG-3Ј and 5Ј-ACTCATCGTACTCCTGCTTG-3Ј; mouse -actin (17), 5Ј-CATCACTATTGGCAACGAGC-3Ј and 5Ј-AC-TCATCGTACTCCTGCTTG-3Ј. ...
To clone a new nuclear receptor, we screened a rabbit heart complementary DNA (cDNA) library with degenerate oligonucleotide probes corresponding to the DNA-binding domain of nuclear receptors, which is highly conserved among receptors. One of the cDNA clones, clone 23, encodes a novel protein of 596 amino acids, and predicted molecular mass is 66 kDa. Homology search analysis identified this protein as rabbit TR4 (TR4-0). We also cloned the cDNA encoding a rabbit TR4 isoform (TR4-1), which lacks the putative C-terminal ligand-binding domain (350 amino acids) caused by a 23-bp exon deletion, which probably occurred during messenger RNA (mRNA) splicing. Northern blot analysis showed that TR4s are expressed with two kinds of mRNAs (9.0 kb and 2.8 kb), both of which are relatively abundant in brain, testis, and bone. RT-PCR analysis, using pairs of primers specific for each TR4, showed that both types of receptor express in various tissues. Furthermore, both are present in primary osteoblasts and bone marrow cells, though the mRNA levels of TR4-0 were much higher than those of TR4-1. A functional study, using a transient transfection assay, showed that both receptors suppressed retinoid X receptor (RXR)-retinoid acid receptor, RXR-TR, and RXR-VDR-mediated transactivation significantly in COS-1 and osteosarcoma cells (UMR-106, ROS17/2.8) and that TR4-0 was much more effective than TR4-1. Unexpectedly, we found that the TR4s effectively suppressed estrogen receptor-mediated transactivation in bone cells, but neither in kidney (COS-1) nor breast cancer cells (MCF-7, one of the major target cells of the estrogen action). Thus, the present study shows a novel property of the TR4 orphan receptor, acting as a bone cell-specific repressor in the estrogen receptor-mediated signaling pathway.
... To minimize the risk of contamination, reagents preparation and PCR master mix, DNA extraction, and electrophoresis were performed in three separate areas. To confirm that amplification inhibitors were not present, a fragment of the human beta-globin gene was tested in all the samples [15]. ...
The aim of this study was to evaluate whether the molecular (kDNA-PCR) and parasitological diagnosis in peripheral blood (PB) could replace the invasive and painful bone marrow collection (BM) in the diagnosis of visceral leishmaniasis (VL). PB from suspected VL patients was evaluated by parasitological and molecular techniques using as the gold standard (GS) a combination of clinical, epidemiological, and immunochromatographic test (PB-rK39) results and parasitological examination of BM. Based on the GS, 38 samples from 32 patients were grouped: Group 1, 20 samples of VL cases, and Group 2, 18 samples of non-VL cases. In order to evaluate the parasitological and molecular techniques in PB, the samples were examined. From Group 1, PB kDNA-PCR was positive in 20 samples and in 19 of 20 in BM kDNA-PCR examination. However, the parasitological examination of buffy coat was insensitive, being able to detect only 4 cases from Group 1. All samples from Group 2 were negative. We concluded that, for the diagnosis of visceral leishmaniasis, the parasitological examination of peripheral blood was not useful; however, molecular diagnosis by kDNA-PCR, performed in peripheral blood, could be useful to replace the parasitological examination of bone marrow.
... Oligonucleotide primers ( Table 2) were designed in the Department of Molecular Biology of Silesian Medical University on the basis of reference sequences (http:// www.ncbi.nlm.nih.gov). Detection of GAPDH gene was performed according Ercolani et al. [15], and b-actin was detected according to Nakajima-Iijima et al. [16]. ...
Aim of the study:
Despite significant progress in the pathology of clear cell renal cell carcinoma (ccRCC), diagnostic and predictive factors of major importance have not been discovered. Some hopes are associated with insulin-like growth factors. The aim of the study was to compare the expression of genes for insulin-like growth factor family in tumours and in tissue of kidneys without cancer.
Material and methods:
Fifty-two patients years with clear cell renal cell cancer were qualified to the study group; patients nephrectomised because of hydronephrosis were included in the control group. Expression of genes were evaluated by RT-PCR.
Results:
Expression of IGFR-1 gene in tumour accounts for about 60% of cases. The incidence is higher than in corresponding adjacent non-cancerous kidney tissues and higher (but with no statistical significance) than in kidney without cancer. Expression of IGFR-2 gene in tumours has not been established. The incidence of the expression in corresponding adjacent non-cancerous kidney tissues is small. Expression of this gene has been present in all specimens from kidneys without cancer. Expression of IGFBP-3 gene ascertained in all (except four) cases of ccRCC and in the majority of clippings from adjacent tissue. It was not found in kidneys from the control group. IGF-1, IGF-2, and IGFR-1 mRNA copy numbers in ccRCC were higher than in the material from the control group.
... -pX1r11R contains a fragment of the 28S gene from the BamHI site at position 1077 to the EcoRI site at position 3603, cloned between the EcoRI and BamHI site of pBR322. -We also used an β-actin probe as an internal con- trol [24]. These fragments of DNA were labeled by random primed synthesis [5] in the presence of [α- 32 P] dCTP (Du Pont, 111 TBq, 3000 Ci/mmol). ...
Ribosomal genes are involved in cellular transcription, translation and gene expression modulation process. An association between 28S/18S rRNA ratio levels with apoptosis and aging has been reported. Moulder et al. [22] and Hashimoto et al. [8] showed an association between apolipoprotein E4 allele and neuronal cell apotosis through diverse mechanisms. The apoE 4 allele is considered a late-onset Alzheimer's disease (AD) risk factor associated with AD pathogenesis. We evaluated the association between apoE4 allele genotyping by PCR and rRNA 28S/18S ratio by slot blotting technique using peripheral blood samples of 18 Alzheimer's disease patients, 18 elderly controls and 18 young controls. A rRNA ratio decrease was observed in AD individuals confirming our previous results but this association is independently of the ApoE4 allele genotype. Thus our results pointed that two different mechanisms are involved in the etiology of Alzheimer disease each one leading independently to cell death. Further studies could investigate these factors.
... The sequences corresponding to these primers are well conserved in CAEV and MVV isolates. Sample DNA integrity was controlled by amplifying the b-actin gene using primers, based on the human sequence [17] ; external – ES30 (5 0 - TCATGT TTG AGA CCT TCA ACA CCC CAG-3 0 ) and ES32 (5 0 -CCA GGG GAA GGC TGG AAG AGT GCC- 3 0 ), and internal – ES31 (5 0 -CCC CAG CCA TGT ACG TTG CTA TCC-3 0 ) and ES33 (5 0 -GCC TCA GGG CAG CGG AAC CGC TCA-3 0 ). PCR reactions were performed with 0.5–1 mg of total DNA in 20 ml of distilled water added to 30 ml of an amplification solution containing: 5 ml of reaction buffer [10Â, 650 mM of Tris–HCl (pH 8.8), 160 mM of (NH 4 ) 2 SO 4 , 0.1% of Tween-20], 5 ml of MgCl 2 (50 mM), 1 ml of dNTP (25 mM of each deoxynucleotide triphosphate: dATP, dGTP, dCTP, dTTP), 0.25 ml of TAQ Polymerase (5 U/ml, EUROBLUETAQ 1 ADN Polymerase-Thermostable, EUROBIO, Les Ulis, France), 0.6 ml of each primer GAG EX3 and GAG EX5 or ES30 and ES32 (33 mM, GIBCO BRL Custom primers—Life Technologies, Grand Island, NY), and 18.15 ml diethyl pyrocarbonate (DEPC)-treated water. ...
... Firstly, in order to track the extraction efficiency and to rule out inhibition of the PCR reaction, the presence of DNA was investigated by analysis of the presence of a cytoplasmic human multicopy ß-actin-gene (Nakajima-Iijima et al. 1985). Negative PCR controls were always included. ...
... 2·1 (Takara Shuzo, Otsu, Japan). The sequences of the primers used in PCR amplification were as follows: 5 -GAG GCA ATG GCC ACC ATG G-3 and 5 -GTA GTC AAA GTC AGA GCA GTC-3 for pS2 (Jakowlew et al. 1984); 5 -GTG GGG GCA AGA TGA AGG TC-3 and 5 -TTA CCC CAA GGG CAC ACC C-3 for insulin-like growth factor (IGF)-binding protein-4 (IGFBP4) (Kiefer et al. 1991b); 5 -TTC GAG AGC AAG TGG AAC CC-3 and 5 -AGC TCC TCC TGA ATG TGG TC-3 for KIAA1051 (Kikuno et al. 1999); 5 -AGC TGT GGA AGC CCT AAC TC-3 and 5 -TCG TAG CCG GTT AAC GCC AG-3 for retinoblastoma-binding protein 8 (Fusco et al. 1998); 5 -AAT GGC GGT TCT CAT GCT GG-3 and 5 -ATC TGG TTG ACT TTG AGC AGG-3 for c-myc promoterbinding protein 1 (Ray & Miller 1991); 5 -ACG AAA AGA GCT ACC GCG AG-3 and 5 -TTG CTG CTG TCG AAG GTG TG-3 for insulin-like growth factor-binding protein-5 (IGFBP5) (Kiefer et al. 1991a); 5 -TGG AGG CAC GGA CCA CTG C-3 and 5 -AGA CAG TCC CCT GCG GTG G-3 for solute carrier family 7 member 5 (Gaugitsch et al. 1992); 5 -GCA CAG AGC CTC GCC TTT G and 5 -CAT CAC GAT GCC AGT GGT A-3 for -actin (Nakajima-Iijima et al. 1985). Twenty-five nanograms of cDNA fragments were labeled with [ -32 P]dCTP using Megaprime DNA labeling systems (Amersham Pharmacia Biotech), and hybridization was carried out using ExpressHyb Hybridization solution (Clontech) according to the manufacturer's instructions. ...
Estrogen plays an important role in many physiological events including carcinogenesis and the development of human breast cancer. However, the molecular mechanisms of estrogen signaling in cancers have not been clarified hitherto and accurate therapeutic prediction of breast cancer is earnestly desired. We first carried out estrogen-responsive expression profiling of approximately 9000 genes in estrogen receptor-positive human MCF-7 breast cancer cells. Based on the results, estrogen-responsive genes were selected for production of a custom-made cDNA microarray. Using a microarray consisting of the narrowed-down gene subset, we first analyzed the time course of the estrogen-responsive gene expression profiles in MCF-7 cells, resulting in subdivision of the genes up-regulated by estrogen into early-responsive and late-responsive genes. The expression patterns of several genes were confirmed by Northern blot analysis. We also analyzed the effects of the estrogen antagonists ICI 182780 and 4-hydroxytamoxifen (OHT) on the estrogen-responsive gene expression profiles in MCF-7 cells. While the regulation of most of the genes by estrogen was completely abolished by ICI 182780, some genes were partially regulated by estrogen even in the presence of OHT. Furthermore, the estrogen-responsive gene expression profiles of twelve cancer cell lines derived from the breast, ovary, stomach and other tissues were obtained and analyzed by hierarchical clustering including the profiles in MCF-7 cells. Several genes also showed up-regulation or down-regulation by estrogen in cell lines other than MCF-7 cells. The significance of the estrogen-responsive genes identified in these analyses concerning the nature of cancer is discussed.
... DNA Probes . DNA fragments used as probes are as follows : Human IL-la cDNA, 2-kb Bam HI fragment of pCDIL-la (11); human IL-lei cDNA, 1-kb Pst I-Acc I fragment of pA-26 (12) (provided by Dr. Webb of Massachusetts Institute of Technology, Cambridge, MA) ; human IL-2 cDNA, 0.8-kb Pvu II-Pst I fragment of pHIG5-3 (13) (provided by Dr. Onoue of Kumamoto University); human IL-3 cDNA, synthetic 31-mer oligonucleotide 5'-GTTGAATGCCTCCAGGTTTGGCCTTCGAAGG-3' corresponding to antisense strand between positions 222 and 252 of the published sequence (14), which is identical to gibbon IL-3 cDNA sequence ; human IL-4 cDNA, 0.3-kb Nhe I-Eco RI fragment (supplied by Ono Pharmaceutical Co., Osaka, Japan) ; human IL-5 cDNA, 1-kb Barn HI fragment ofph-IL-5-30 (15); human IL-6 cDNA, 1.1 kb Bam HI-Eco RI fragment of pBSF2-38 (16) (provided by Dr. T. Hirano of Osaka University); /3-actin gene, 0.4-kb Hinf I fragment which encodes the 4th exon and its 3' intron (17). These fragments, except for human IL-3 synthetic oligonucleotide, were labeled to obtain the specific activity of 600-1,200 cpm/pg using random primers (18) and a-[ 32P]dCTP (3,000 Ci/mmol, Amersham Corp., Arlington Heights, IL). ...
We have analyzed expression patterns of 7 lymphokine mRNAs by Northern blot analyses in 19 different human T cell clones derived from patients with adult T cell leukemia. However, we were not able to reveal particular combinations of lymphokine production that allowed classification of human T cells. Especially, four clonally related leukemic lines that were established independently from the same patient with adult T cell leukemia expressed different combinations of lymphokine mRNAs, indicating that the expression of various lymphokines is not fixed but rather variable even among progenies of a single T cell clone.
... The resulting cDNA was then subjected to 30 cycles of PCR for HGF, c-Met, and -actin using a DNA thermal cycler (Perkin Elmer Cetus, Norwalk, CT), Taq DNA polymerase (Takara, Tokyo, Japan), and primers specific for human HGF, c-Met, or -actin. The primer sequences were designed as follows: for HGF primers (primers 1 and 2), 5'-CCATGAATTTGACCT-CTATG (472-492) and 5'-ACTGAGGAATGTCACGACT (750-731); for c-Met primers (primers 3 and 4), 5'-AGAT-GAACGTGAACATGAAG (295-314) and 5'-CTAAT-GAGTTGATCATAG (589-568) [13]; for -actin primers (primers 5 and 6), 5'-CTCACCATGGATGATGATAT and 5'-TGGGTCATCTT-CTCGCGGTT [14]. Each primer gives a fragment length of 278 base pairs (bp) for HGF, 294 bp for c-Met, and 368 bp for 3-actin. ...
Human endometrial regeneration has been considered to be modulated by several growth factors. However, little is known about the detailed mechanisms involved in the repair of the en-dometrium during the menstrual period. Hepatocyte growth factor (HGF) is a pleiotropic growth factor that reportedly acts on various epithelial cells. In the present study, we observed HGF and mRNA expression in human endometrium and mRNA expression in cultured endometrial epithelial cells of the c-met gene by reverse transcription-polymerase chain reaction and Southern blot hybridization. We also examined the biological role of HGF on the regeneration of the endometrium by using cultured endometrial epithelial cells in their proliferative phase. With the proliferation assay, the addition of 10-50 ng/ml of HGF showed that HGF was mitogenic in a dose-dependent manner. With Boyden's chamber technique, 50 ng/ml of HGF significantly stimulated cell migration. In a three-dimensional cell-culture system, the endometrial epithelial cells formed cell clusters and gradually formed epithelial lumens, both of which were stimulated by 50 ng/ml of HGF. Results suggest that HGF stimulates the proliferation and migration of, and morphogenic changes in, endometrial epithelial cells. HGF may modulate the regeneration of the endometrium during menstruation.
Amplification and increased expression of the mdr1 gene associated with multidrug resistance in human tumors were found in multidrug-resistant sublines of human myelogenous leukemia K562 selected with vincristine (K562/VCR) or adriamycin (K562/ADM). In two revertant cell lines of K562/ADM, amplification of the mdr1 gene was maintained at the same level as in K562/ADM, but expression of the 4.5-kilobase mdr1 mRNA was greatly decreased, indicating that amplified genes may be inactivated at the level of transcription without a corresponding loss of amplified DNA.
We previously isolated a Fisher rat fibroblast mutant, B812, that has the unique property of temperature-dependent transformation by various oncogenic retroviruses. At the permissive temperature (35 degrees C), this mutant was sensitive to oncogenic transformation and formed foci on a dish at the same frequency as did the parental fibroblast cell line. When Kirsten murine sarcoma virus (Ki-MSV) was applied to the cells, the frequency of focus formation decreased more than 25-fold at the nonpermissive temperature (39 degrees C), whereas the cells expressed nearly the same level of the ras transcript as well as the ras protein. The temperature-restricted focus formation was fully reversible and was completely suppressed upon fusion with the wild-type parent cell. In addition to ras, the mos, fos, src, and erbB-2 oncogenes transformed this mutant with the same temperature dependence as described above; polyomavirus middle T antigen, adenovirus type 12, and human papillomavirus 16-E67 also transformed, but without temperature dependence. These results suggest that ras, fos, mos, src, and erbB-2 use a common cellular pathway for transforming cells.
Recently it was demonstrated that beta-actin can be produced in Saccharomyces cerevisiae by using the expression plasmid pY beta actin (R. Karlsson, Gene 68:249-258, 1988), and several site-specific mutants are now being produced in a protein engineering study. To establish a system with which recombinant actin mutants can be tested in vivo and thus enable a correlation to be made with functional effects observed in vitro, a yeast strain lacking endogenous yeast actin and expressing exclusively beta-actin was constructed. This strain is viable but has an altered morphology and a slow-growth phenotype and is temperature sensitive to the point of lethality at 37 degrees C.
Recombinant phages that carry the human smooth muscle (enteric type) gamma-actin gene were isolated from human genomic DNA libraries. The amino acid sequence deduced from the nucleotide sequence matches those of cDNAs but differs from the protein sequence previously reported at one amino acid position, codon 359. The gene containing one 5' untranslated exon and eight coding exons extends for 27 kb on human chromosome 2. The intron between codons 84 and 85 (site 3) is unique to the two smooth muscle actin genes. In the 5' flanking region, there are several CArG boxes and E boxes, which are regulatory elements in some muscle-specific genes. Hybridization with the 3' untranslated region, which is specific for the human smooth muscle gamma-actin gene, suggests the single gene in the human genome and specific expressions in enteric and aortic tissues. From characterized molecular structures of the six human actin isoform genes, we propose a hypothesis of evolutionary pathway of the actin gene family. A presumed ancestral actin gene had introns at least sites 1, 2, and 4 through 8. Cytoplasmic actin genes may have directly evolved from it through loss of introns at sites 5 and 6. However, through duplication of the ancestral actin gene with substitutions of many amino acids, a prototype of muscle actin genes had been created. Subsequently, striated muscle actin and smooth muscle actin genes may have evolved from this prototype by loss of an intron at site 4 and acquisition of a new intron at site 3, respectively.
The accumulation of the cytoskeletal beta- and gamma-actin mRNAs was determined in a variety of mouse tissues and organs. The beta-isoform is always expressed in excess of the gamma-isoform. However, the molar ratio of beta- to gamma-actin mRNA varies from 1.7 in kidney and testis to 12 in sarcomeric muscle to 114 in liver. We conclude that, whereas the cytoskeletal beta- and gamma-actins are truly coexpressed, their mRNA levels are subject to differential regulation between different cell types. The human gamma-actin gene has been cloned and sequenced, and its chromosome location has been determined. The gene is located on human chromosome 17, unlike beta-actin which is on chromosome 7. Thus, if these genes are also unlinked in the mouse, the coexpression of the beta- and gamma-actin genes in rodent tissues cannot be determined by gene linkage. Comparison of the human beta- and gamma-actin genes reveals that noncoding sequences in the 5'-flanking region and in intron III have been conserved since the duplication that gave rise to these two genes. In contrast, there are sequences in intron III and the 3'-untranslated region which are not present in the beta-actin gene but are conserved between the human gamma-actin and the Xenopus borealis type 1 actin genes. Such conserved noncoding sequences may contribute to the coexpression of beta- and gamma-actin or to the unique regulation and function of the gamma-actin gene. Finally, we demonstrate that the human gamma-actin gene is expressed after introduction into mouse L cells and C2 myoblasts and that, upon fusion of C2 cells to form myotubes, the human gamma-actin gene is appropriately regulated.
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.
An enhancer of the human beta-actin gene and a factor that specifically interacts with it were detected. A mobility shift assay showed that the factor bound to the 25-base-pair sequence (between +759 and +783 downstream from the cap site) with high specificity. This finding correlated with those of DNase I protection and exonuclease III digestion assays. This binding region of the beta-actin enhancer contained a hyphenated dyad symmetry and an enhancer core-like sequence. In vitro competition experiments indicated that the factor did not bind to the simian virus 40 enhancer core region.
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.
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.
Although β actin mRNA is down-regulated during myogenesis, the β actin promoter confers constitutive expression when joined to heterologous genes transfected into a variety of different cell backgrounds, including differentiated muscle. Normal promoter activity is dependent upon the binding of a ubiquitous factor to the CCAAT-box element. Loss or reduction in factor binding correlates with a major reduction in promoter activity both in vivo and in vitro. The binding domain covers approximately 23 base pairs as determined by DNase footprinting. Methylation of A and G residues in and adjacent to the CCAAT box results in the loss of factor binding. Mutations across the binding domain indicate that the sequence GCCAATCAG within the domain is sufficient as a recognition sequence for factor binding. This binding is not competed by the α cardiac actin CCAAT sequence. Bandshift experiments demonstrate a predominant single band of similar mobility in nuclear extracts from various cells and tissues, with the exception of HeLa cells. The prevalence of the factor and its recognition sequence in a variety of promoters suggests that this factor has a common role in the transcriptional activation of several eukaryotic promoters.
The effects of phorbol ester (TPA) and other known stimulators such as tumor necrosis factor (TNF), interleukin-1, and lipopolysaccharide on induction of mRNA for manganese-superoxide dismutase (Mn-SOD) were investigated in various cell lines. TPA enhanced Mn-SOD mRNA expression in TNF-resistant cell lines including HeLa cells, in which the other reagents also induced expression of the gene, but did not affect TNF-sensitive cells, in which the other stimulators did not alter expression of the gene. HeLa cells which had been desensitized to TPA by pretreatment with TPA for 24 h expressed Mn-SOD mRNA at a slightly higher level than the cells without TPA treatment. TPA-pretreated cells stimulated with TNF, however, expressed Mn-SOD mRNA at about twice the level of TNF-stimulated, TPA-untreated cells. When protein synthesis was inhibited by cycloheximide during TPA pretreatment, TNF no more enhanced the Mn-SOD mRNA accumulation. These data suggest that at least two separate signal-transducing pathways are involved in expression of this gene. One is triggered by protein kinase C activation itself in the absence of new protein synthesis. The other can be activated by stimulation with TNF, interleukin-1, or lipopolysaccharide and in which a protein factor that can be induced by TPA treatment is involved.
Androgens are essential for the growth and differentiation of the prostate. Androgen signalling is mediated by the androgen receptor (AR), a ligand-dependent transcription factor. Androgen ablation is the standard treatment for prostate cancer but nearly all patients relapse with androgen-independent disease. Progression of prostate cancer is often associated with changes in the AR-signalling pathway. The project aimed to investigate molecular mechanisms underlying the regulation of AR gene expression in hormone-relapsed prostate cancer. An inducible and a constitutive gene expression system were used to overexpress AR in human prostate cancer cell lines. The TetOff inducible gene expression system, which offers the advantage of quantitatively regulating AR gene expression in response to varying concentrations of tetracycline, was used. A highly tTA-expressing stable ceil line (DUTetOff) was established in the AR- negative DU145 cell line, and a functional AR expression vector (pTRE-AR) was constructed. Transient assays with pTRE-AR in DUTetOff, DU145, PC-3, DUSF and COS-1 cells indicated that, while AR mRNA was expressed in all cells tested, the AR transcript was not translated in DUTetOff and DU145 cells. To develop a constitutive AR gene expression system, the full-length human AR cDNA was introduced into a DU145-derived serum-free subline (termed DUSF). Stable clones were screened for AR expression by immunocytochemistry, Western analysis and RT-PCR. Up- regulation of AR mRNA and protein was detected in DUSF transfectants following androgen treatment. Endogenous PSA mRNA expression was observed in untransfected DUSF cells, while androgen treatment of the transfectants implied an AR- and androgen-independent mechanism for PSA regulation. The work described in this thesis indicates that overexpression of AR in AR-negative cells permits androgen-mediated AR gene expression, and implies an alternative mechanism for PSA activation. The stable AR-expressing DUSF cells provide a useful model system for investigating androgen-independent regulatory elements involved in PSA gene regulation and, elucidating the mechanisms involved in prostate cancer progression.
Primary ciliary dyskinesia (PCD) is the term used to encompass the diseases known as Kartagener syndrome (OMIM 244000) and immotile cilia syndrome (OMIM 242650/242680/242670). PCD is an autosomal recessive disease with an estimated prevalence of 1 in 20,000. The main clinical features of PCD are recurrent sinopulmonary infections as a direct consequence of a primary abnormality of cilia. Cilia are highly complex organelles and this has led to the hypothesis that mutations in a number of different genes may lead to the PCD phenotype. At the time that this research project was begun none of the disease genes causing PCD had been identified. The aim of this project was to map and clone the gene(s) for PCD using a positional cloning strategy. This thesis describes the results of two genome screens; the first genome screen used the technique of homozygosity mapping in a large consanguineous German family. This highlighted 3 areas of interest which were then further evaluated in families that shared an identical ultrastructural phenotype. The results did not achieve statistical significance but suggested potential loci for PCD on chromosome 17q and chromosome 11q. The second genome screen was performed in individuals from the isolated community of the Faroe Islands. This screen revealed an area of interest on chromosome 16p. This area continues to be evaluated. During the course of this thesis a mutation in a gene that codes for an intermediate dynein (IC78) (Pennarun, Escudier et al. 1999) was identified and significant evidence for linkage was also found on chromosome 19q. (Meeks, Walne et al. 2000). In summary PCD is a genetically heterogeneous disease. Two loci have been published (Pennarun, Escudier et al. 1999) (Meeks, Walne et al. 2000) and at least two further potential loci have been identified as part of this thesis.
Pre-eclampsia is a disorder affecting 5 to 10% of all pregnancies. In the pre-eclamptic placenta, increased levels of an inositolphosphoglycan (IPG) second messengers have been reported. The objective of this thesis has been the study of IPGs in the placenta and their possible release by the glycosylphosphatidylinositol-phospholipase D (GPI-PLD) enzyme. The presence of IPGs was investigated in the placenta. Immunostaining on sections of chorionic villi revealed the presence of IPGs and confirmed the higher levels of IPGs in pre-eclampsia. IPGs of both sub-types A and P were extracted from microvilli preparations of both normal and pre-eclamptic placentae. After showing the presence of biologically active IPGs in microvilli preparation, the next step was to investigate the presence of glycosylphosphatidylinositol (GPI), the putative precursor molecule of IPGs. GPI was extracted from microvilli of normal placenta but unexpectedly not detected in pre-eclamptic samples. An elevated GPI-PLD activity could cause increased catabolism of GPI, and thus be responsible for both the absence of detectable GPI and the increased IPG levels found in pre-eclamptic samples. GPI-PLD hydrolysis activity was measured in microvilli preparations from both normal and pre-eclamptic placenta. Whereas GPI-PLD is described as a 100 kDa protein, a protein of 50 kDa was mainly detected. GPI-PLD mRNA was not detected in the placenta. We therefore propose a model in which placental GPI-PLD is taken up by the syncytiotrophoblast from the maternal blood and transferred to a lysosomal compartment where it is cleaved into an active 50 kDa protein before returning to the plasma membrane. Finally, the susceptibility of extracted placental GPI to hydrolysis by recombinant GPI-PLD has been studied. GPI-PLD was able to cleave GPI in vitro. To conclude, this work confirms the presence of IPGs in placenta and more specifically in microvilli and supports previous reports of increased IPG-P type in preeclampsia. The study of placental GPI and GPI-PLD throws light on the possible mechanism underlying the generation of IPGs in both normal and pre-eclamptic microvilli.
ErbB4 is the fourth and, to date, final member of the ErbB family of receptor tyrosine kinases, which includes the EGF receptor. ErbB receptors are expressed in many tissues during development, and thought to have roles in proliferation, survival, and differentiation. Additionally, their expression is deregulated in many tumour types. Mice have previously been created which lack the ErbB4 receptor. These show defects in cranial neural crest cell migration and axon pathfinding, but die by embryonic day (E) 10.5 due to failure of trabeculation to occur in the heart. To circumvent this mid-embryonic lethality and to allow the study of later genotypes, I have rescued the cardiac defect in ErbB4-/- mice using a construct expressing human ErbB4 (HER4) under a cardiac-specific promoter. Expression of this construct is shown to be specific to the heart. ErbB4-/- HER4heart mice survive to adulthood and appear superficially normal. However, fewer ErbB4-/- rescued pups are born than predicted by Mendelian genetics. Additionally, ErbB4-/- HER4heart mothers fail to rear most of their pups, probably due to a lactation defect. I demonstrate that ErbB4-/- HER4heart breast fails to differentiate correctly during pregnancy, and also that ErbB4 regulates phosphorylation of Stat5, a central molecule in milk production. The ErbB4-/- neural crest and cranial nerve phenotype is shown to be replicated by ErbB4-/- HER4heart embryos, and an aberrant nerve linking the trigeminal and facial ganglia persists at E17.5. I also show results of a separate study into the roles of sulphated proteoglycans in wild type and ErbB4-/- embryonic hindbrain regions.
The extracellular matrix metalloproteases (MMPs) secreted by various human tumor cells play a crucial role in tumor cell invasion and metastasis, but their expression in malignant mesothelioma (MM) cells has not been examined. In this study, we have investigated the spectrum of MMPs and tissue inhibitors of metalloproteases (TIMPs) produced by 8 MM cell lines. Using RT‐PCR, we found that all investigated MM cell lines expressed genes encoding mRNA for MMP‐1 (interstitial collagenase), MMP‐2 (gelatinase A), MMP‐3 (stromelysin‐1), MMP‐9 (gelatinase B) and TIMPs 1, 2 and 3. We also found that 6/8 MM cell lines expressed MMP‐7 (matrilysin) and 3/8 MM cell lines expressed MMP‐10 (stromelysin‐2). MMP‐11 (stromelysin‐3) was not detected in any of the MM cell lines. Production of MMP‐2 and MMP‐9 was confirmed using gelatin zymography. In addition, all MM cell lines secreted a 66 kDa metalloprotease, while 3/8 MM cell lines secreted 46, 48, 51 and 63 kDa metalloproteases which specifically degraded the extracellular matrix components fibronectin, vitronectin and laminin. The 66 kDa protease was identified as MMP‐3 by Western blot. Our results reveal a broad spectrum of MMPs and TIMPs produced by MM cells and indicate that different substrate specificities of MMPs may play a role in MM cell invasion. © 2001 Wiley‐Liss, Inc.
Granule major basic protein (MBP) is expressed exclusively in eosinophils, basophils, and placental trophoblasts. To identify thecis-elements and transcription factors involved in regulating MBP expression, we subcloned 3.2 kb of sequence upstream of the exon 9 transcriptional start site (P2 promoter) and serial 5′ deletions into the pXP2 luciferase reporter vector. An 80% decrement in promoter activity was obtained when MBP sequences between bp −117 to −67 were deleted. To identify transcription factors that bind to and transactivate through the bp −117 to −67 region, we first compared the upstream genomic sequences of human and murine MBP; a potential GATA binding consensus site was conserved in the 50-bp region between the two genes. To determine which GATA proteins bind this consensus site, we performed electrophoretic mobility shift assays (EMSAs), which showed that both GATA-1 and GATA-2 can bind to this consensus site. To determine the functionality of this site, we tested whether GATA-1 and GATA-2, either individually or in combination, can transactivate the MBP promoter in the Jurkat T cell line. Cotransfection with a GATA-1 expression vector produced 20-fold augmentation of MBP promoter activity, whereas GATA-2 had no activity. In contrast, combined cotransfection of GATA-1 and GATA-2 decreased the ability of GATA-1 to transactivate the MBP promoter by approximately 50%. Our results provide the first evidence for a GATA-1 target gene in eosinophils, a negative regulatory role for GATA-2 in MBP expression, and possibly eosinophil gene transcription in general during myelopoiesis.
Gender differences in vascular thromboses are well known, and there is evidence that platelets may be involved in these differences and that sex hormones affect platelet function. We characterized the expression of the estrogen receptor (ER ), estrogen receptor β (ER β), progesterone receptor (PR), and androgen receptor (AR) in the megakaryocyte lineage. Megakaryocytes generated ex vivo from normal human CD34+ stem cells contained RNA for ER β and AR, which increased with cell differentiation. Platelets and human erythroleukemia (HEL) cells also contained ER β and AR transcripts. No ER or PR messenger RNA or protein was detected in the megakaryocyte lineage. Immunofluorescence microscopy showed that ER β protein was present in glycoprotein (GP) IIb+ megakaryocytes and the HEL megakaryocytic cell line in a predominantly cytoplasmic location. AR showed a cytoplasmic and nuclear distribution in GPIIb+ and GPIIb− cells derived from CD34+ cells and in HEL cells. Western immunoblotting confirmed the presence of ER β and AR in platelets. Megakaryocyte and HEL AR expression was up-regulated by 1, 5, and 10 nmol/L testosterone, but down-regulated by 100 nmol/L testosterone. These findings indicate a regulated ability of megakaryocytes to respond to testosterone and suggest a potential mechanism through which sex hormones may mediate gender differences in platelet function and thrombotic diseases.
The flt3 ligand (FL) is a growth factor for primitive hematopoietic cells. Serum levels of FL are inversely related to the number and proliferative capacity of early hematopoietic progenitors. We sought to elucidate the molecular mechanism underlying this regulation. Expression of FL was examined in peripheral blood (PB) and bone marrow (BM) cells under normal steady-state hematopoiesis and during transient BM failure induced by chemoradiotherapy in 16 patients with hematological malignancies. Using anti-FL antibodies in Western analysis, flow cytometry, and confocal microscopy, we detected high levels of preformed FL inside but not on the surface of T lymphocytes in steady-state hematopoiesis. Intracellular FL colocalized with giantin and ERGIC-53, indicating that it is stored within and close to the Golgi apparatus. After chemotherapy-induced hematopoietic failure, FL rapidly translocated to the surface of T lymphocytes and the levels of FL released to serum increased approximately 100-fold. Expression of FL mRNA was enhanced only about sevenfold; a similar, twofold to sixfold increase in mRNA was observed in the thymus and BM of mice with irradiation-induced aplasia. Upregulation of FL mRNA was delayed when compared with the appearance of cell surface-associated and soluble protein isoforms. The described changes in FL expression in response to chemotherapy-induced aplasia were observed in all patients, irrespective of the diagnosis and treatment regimen. Our data demonstrate that mobilization of preformed FL from intracellular stores rather than de novo synthesis is responsible for increased FL levels in BM failure.
Extracellular signal-regulated kinase (ERK) is an important intermediate in signal transduction pathways that are initiated by many types of cell surface receptors. It is thought to play a pivotal role in integrating and transmitting transmembrane signals required for growth and differentiation. Constitutive activation of ERK in fibroblasts elicits oncogenic transformation, and recently, constitutive activation of ERK has been observed in some human malignancies, including acute leukemia. However, mechanisms underlying constitutive activation of ERK have not been well characterized. In this study, we examined the activation of ERK in 79 human acute leukemia samples and attempted to find factors contributing to constitutive ERK activation. First, we showed that ERK and MEK were constitutively activated in acute leukemias by in vitro kinase assay and immunoblot analysis. However, in only one half of the studied samples, the pattern of ERK activation was similar to that of MEK activation. Next, by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblot analysis, we showed hyperexpression of ERK in a majority of acute leukemias. In 17 of 26 cases (65.4%) analyzed by immunoblot, the pattern of ERK expression was similar to that of ERK activation. The fact of constitutive activation of ERK in acute leukemias suggested to us the possibility of an abnormal downregulation mechanism of ERK. Therefore, we examined PAC1, a specific ERK phosphatase predominantly expressed in hematopoietic tissue and known to be upregulated at the transcription level in response to ERK activation. Interestingly, in our study, PAC1 gene expression in acute leukemias showing constitutive ERK activation was significantly lower than that in unstimulated, normal bone marrow (BM) samples showing minimal or no ERK activation (P = .002). Also, a significant correlation was observed between PAC1 downregulation and phosphorylation of ERK in acute leukemias (P= .002). Finally, by further analysis of 26 cases, we showed that a complementary role of MEK activation, ERK hyperexpression, and PAC1 downregulation could contribute to determining the constitutive activation of ERK in acute leukemia. Our results suggest that ERK is constitutively activated in a majority of acute leukemias, and in addition to the activation of MEK, the hyperexpression of ERK and downregulation of PAC1 also contribute to constitutive ERK activation in acute leukemias.
Myocarditis, a life threatening disease, is still not adequately treated. Histamine plays an important role in physiology and pathophysiology of cardiovascular system. All four histamine receptors (H1R - H4R), are present in the heart. Experimental autoimmune myocarditis (EAM) was used to investigate which histamine receptor had a greater impact on the disease's progression. EAM was evoked in Lewis rats by porcine myosin immunization. Mepyramine, ranitidine and ciproxifan were used to inhibit H1R, H2R and H3R receptors, respectively, and 2,4-diaminopyrimidines: ST994, ST1012, ST1006 were ligands of H4R. Quinapril, an ACE inhibitor, served as a reference drug. Drugs were administered daily, either from 0 - 2 weeks or from 2 to 4 weeks post EAM induction. Cardiac dysfunction developed with significant decreases in left ventricular ejection fraction and fractional shortening due to dilatation and wall thickening. EAM rats treated with mepyramine and ST994 in weeks 0 - 2 had the lowest decreases. These treated with ST994, ST1012 or quinapril performed much better the following 2 weeks without therapy than did the other groups. On autopsy their hearts were smaller, less fibrotic, histopathological changes in them of a lower grade. When the treatment started with 2 weeks' delay, the ST994-treated EAM rats showed the highest median survival. H4 receptor antagonism inhibits heart remodelling, preserves heart contractility, improves survival and may be of potent therapeutic relevance in human clinics. The blockade of H1 receptor inhibits heart dilatation but does not prolong the life.
Proteomic methods allow detecting breed-specific differences at protein level. The objective of the present study was to compare muscle proteome profiles of Hungarian Merino and Tsigai sheep breeds by two dimensional gel electrophoresis and mass spectrometry based protein identification. Approximately 327 protein spots were detected in musculus longissimus dorsi and 14 protein spots, identified as 12 proteins, showed significant differences in their intensity (P < 0.05) between breeds. Seven and five proteins had higher expression in Merino and Tsigai skeletal muscle, respectively. Identified proteins were classified as structural, carbohydrate metabolism-related and miscellaneous ones. Six genes, which represent the three protein groups by function, were analysed by quantitative real-time PCR. All of the identified structural proteins have shown higher intensity in Merino, while the expression of identified glycolytic enzymes and myoglobin were higher in Tsigai lambs. One of the structural and one of the carbohydrate metabolism-related protein showed differences in expression at mRNA level by quantitative real-time PCR, as well. To the best of our knowledge, it was the first differential proteome analysis of sheep muscle and these results contribute to a better understanding of molecular differences between breeds.
Real-time RT-PCR using a TaqMan® fluorogenic detection system is a simple and sensitive assay for quantitative analysis of gene transcription. This method is of potential usefulness in quantifying mRNA of a target gene in autopsy material that has undergone only a small amount of postmortem degradation. The TaqMan fluorogenic detection system can monitor PCR in real time using a dual-labeled fluorogenic hybridization probe (TaqMan probe) and a polymerase with 5′–3′ exonuclease activity. The procedures of the quantitative RT-PCR are as follows: RNA is extracted from autopsy material and used to synthesize cDNA by an RT reaction, and the target of interest is amplified and detected by the real-time PCR. The absolute amount of target mRNA in the sample is then determined relative to a standard curve. This chapter describes the methodology of the TaqMan fluorogenic detection system in handling autopsy material in the gene transcription assay.
Recent studies suggest that the renal tubule is not only a target of endothelin-1 (ET-1) but is also a possible source of endothelin. Thus, we attempted to detect specific preproET-1 messenger RNA (mRNA) as well as immunoreactive ET-1 secretion in renal epithelial cell lines. Madin-Darby canine kidney (MDCK) cells expressed a single 2.3 kb preproET-1 mRNA. In medium conditioned by MDCK for 24 hours there was a significant amount of immunoreactive ET-1 detected by sandwich type immunoassay. Both mRNA expression and mature ET-1 secretion were stimulated by an addition of transforming growth factor (TGF)-β to the basolateral side in a time-dependent manner, but the increase in peptide secretion lagged behind ET-1 mRNA expression by several hours. The basal secretion of ET-1 was polarized, with 2.3-fold greater secretion into the basolateral side over the apical side, while the difference was augmented to 7.5-fold by TGF-β. In contrast, LLCPK1 (pig kidney) cells secreted little ET-1 associated with a lower level of ET-1 mRNA expression. Regarding the effect of ET-1, it elicited [Ca2+]i transients in LLC-PK1 but not in MDCK. These data clearly show that renal tubules can express preproET-1 mRNA, synthesize mature ET-1, and secrete it preferentially to the basolateral side of the tubule cells, all of which are enhanced by TGF-β. The data suggest that ET- l produced by the renal tubule may function as a paracrine hormone in regulating tubular functions.
Object
Quantitative and qualitative alterations in the epidermal growth factor receptor (EGFR) commonly occur in many cancers in humans, including malignant gliomas. The aim of the current study was to evaluate molecular and cellular effects of OSI-774, a novel EGFR tyrosine kinase inhibitor, on nine glioblastoma multiforme (GBM) cell lines.
Methods
The effects of OSI-774 on expression of EGFR messenger (m)RNA and protein, proliferation, anchorage-independent growth, and apoptosis were examined using semiquantitative reverse transcription–polymerase chain reaction, immunocytochemical analysis, Coulter counting, soft agar cloning, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling/fluorescence-activated cell sorting, respectively. All p53 genes were completely and bidirectionally sequenced.
Suppression of anchorage-independent growth by OSI-774 was inversely correlated to the induction of EGFR mRNA during relative serum starvation (r = −0.74) and was unrelated to p53 status. Overall, suppression of anchorage-independent growth was a considerably stronger effect of OSI-774 than inhibition of proliferation. The extent of OSI-774–induced apoptosis positively correlated with both proliferation and anchorage-independent growth of GBM cell lines (r = 0.75 and 0.79, respectively). In a single cell line derived from a secondary GBM, exposure to concentrations of greater than or equal to 1 μmol/L resulted in a substantial net cell loss during proliferation studies.
Conclusions
The induction of EGFR mRNA may constitute a cellular mechanism to counteract the inhibitory effect of OSI-774 on the anchorage-independent growth of GBM cells. In contrast, no considerable correlation could be established between baseline expression levels of EGFR (both mRNA and protein) in GBM cell lines and their biological response to OSI-774. The OSI-774 induced greater ( p53 -independent) apoptosis in more malignant GBM phenotypes and may be a promising therapeutic agent against secondary GBM.
To investigate whether circulating thyroglobulin (Tg) messenger ribonucleic acid (mRNA) and sodium/iodide symporter (NIS) mRNA transcripts in peripheral blood are valuable in the follow-up of patients with thyroid cancer, we developed highly sensitive nested Tg and NIS mRNA detection assays and compared their accuracy with serum thyroglobulin (sTg) and whole body scan with ¹³¹I during the monitoring of 34 patients with well differentiated thyroid carcinoma who had undergone total thyroidectomy (17 of 34 also submitted to thyroid ablation with radioiodine) and were taking T4.
Circulating Tg mRNA was found in 13 of 34 patients, 5 of 13 with detectable and 8 of 13 with undetectable sTg. From these 8 patients with undetectable Tg, 6 showed no cervical radioiodine uptake, and 3 presented proven metastatic disease (2 of them positive for antithyroglobulin antibodies). NIS mRNA was detected in 11 of 34 patients, but its measurement did not improve the ability to detect patients with metastases. Overall, identification of metastatic thyroid cancer was better associated with Tg mRNA than with NIS mRNA, sTg, or whole body scan (83% vs. 16.6% vs. 50% vs. 50%; P < 0.001).
These data showed that circulating Tg mRNA is not only a more sensitive marker of residual thyroid tissue or thyroid cancer than sTg, particularly in patients during T4 therapy and with positive antithyroglobulin antibodies, but also was more sensitive than NIS mRNA in all patients.
Expression of nm23 messenger RNA was investigated by primer extension-reverse transcription and polymerase chain reaction method (RT-PCR) in patients with hepatocellular carcinomas (HCC). Relative density units (RDU) of the expression of nm23 (Mean ± S.D.) in non-cirrhotic liver, cirrhotic liver and HCC were 0.86 ± 0.26, 0.84 ± 0.24 and 0.56 ± 0.38, respectively. RDU in solitary HCCs less than 5 cm in diameter with no portal thrombus or capsular infiltration and multinodular HCCs in the whole liver were 0.74 ± 0.51 and 0.38 ± 0.26 (p<0.05). Two years after gross resection of solitary HCCs, RDU in recurrent cases and in recurrence-free cases were 0.95 ± 0.57 and 0.49 ± 0.30, respectively (p<0.05). These results suggest that the expression of nm23 decreases during progression of HCC. It may be a useful factor for predicting the risk of intrahepatic metastasis from HCC in individual cases at the time of initial surgical treatment.
HO-1 (HSP32) is oxidative stress-inducible (Maines, 1997) and catalyzes oxidation of heme to biologically active molecules: iron, a gene regulator, biliverdin, an antioxidant and carbon monoxide. Consecutive downstream mediation is involved in vasodilation, stimulation of guanylate cyclase, and neuronal transmission (Hartsfiled et al., 1997).
The contractile elements of striated vertebrate skeletal muscle, the myofibrils, contain thin filaments, which are 6 nm in diameter and consist mainly of actin, and thicker myosin filaments with a diameter of 16 nm (Fig. 10.1). During muscle contraction, the filaments undergo a sliding movement relative to each other (sliding filament mechanism). This is brought about by the reversible formation of bridges between the myosin molecules and the actin filaments, which bind, change their conformation and then dissociate (bridge cycle). The required energy is supplied by the hydrolysis of ATP. The sliding distance (step size) per molecule of ATP hydrolysed is controversial; the most recent measurements give a value of 40 nm [102, 286]. As yet, the molecular processes of bridge formation and change in conformation have not been fully defined [79, 257, 267]. Because one ATP is hydrolysed per bridge cycle, the ATPase activity can be used as an in vitro indication of contraction. Isolated myosin has a very low ATPase activity, but this increases up to 1000-fold after combination with actin to give actomyosin. ATPase is inhibited in relaxed muscle; the relief of ATPase inhibition and the triggering of contraction in activated muscle involves an increase in the sarcoplasmic Ca2+ concentration from about 0.1 μmol/1 at rest to about 10 μmol/l on activation. The calcium-dependent regulation of contraction is brought about by tropomyosin and the troponin complex, which are an integral part of the thin filaments [238].
Resting platelets are small discoid cells that are capable, upon appropriate stimulus, of undergoing remarkable changes in morphology and activity. In vitro, these include an initial shape change to a spherical form, with the extension of pseudopodia in response to low concentrations of activating agents, incubation in the cold, or mechanical activation. This shape change may be reversed by incubation at 37°C, indicating that no major irreversible reorganization has taken place within the cell. Further activation leads to the extension of numerous long pseudopodia, followed by aggregation and the secretion of the physiologically active contents of the granules. Finally, aggregated platelets express contractile activity in causing the retraction of an aggregate or fibrin clot. An alternative method of studying in vitro activation is to allow platelets to attach to surfaces such as glass, where again initially they extend pseudopodia and finally spread to a flattened form resembling a fried egg. There are a wide variety of stimuli that can cause these reactions; these include collagen, thrombin, ADP, serotonin, and the Ca2+ ionophore A23187; their effects have been reviewed extensively by Siess (1989). The physiological counterparts of these in vitro activities are an initial activation by subendothelial collagen, exposed by damage to the endothelium, followed by further activation induced by the formation of thrombin and augmented by positive feedback from the release of ADP and serotonin from platelet degranulation. In physical terms, the platelets attach to the exposed subendothelium, form a hemostatic plug, and finally cause clot retraction to close the lesion.
Expression of CD44 and its variants is associated with clinically aggressive behavior of some human cancers. The present study was undertaken to determine the expression level of these CD44 mRNAs in relation to the clinicopathologic features and prognosis of gastric cancer. Using reverse transcription polymerase chain reaction followed by Southern blotting, we examined the expression of the standard and variant (v6 and v9) forms of CD44 mRNA in 73 cases of gastric cancer. We determined the ratio of mRNA expression in cancer tissue to normal tissue (T/N ratio) and evaluated the correlations of the ratio with clinico-pathologic features, tumor progression and prognosis. The expression level of the standard form of CD44 (CD44s) mRNA correlated with peritoneal dissemination only, and that of CD44v9 mRNA did not significantly correlate with any clinicopathologic factor. The expression level of CD44v6 mRNA was significantly higher in patients with lymph node metastasis and liver metastasis. In 48 curatively resected patients, the expression level of CD44v6 mRNA correlated with the site of recurrence. Furthermore, there was a significant survival advantage in patients with low expression of CD44v6 mRNA compared with those with high expression. The level of CD44v6 mRNA expression may be a potential prognostic indicator and may he useful as a predictor for distant metastasis and recurrence in patients with gastric cancer. (C) 1998 Wiley-Liss, Inc.
The human placenta is capable of producing a variety of haematopoietic growth factors in vitro. It is not clear, however, whether the placenta produces such factors in vivo and if so, whether placental production of haematopoietic growth factors has a physiological role in fetal haematopoietic development. As a step toward making this determination, we assessed whether the onset of placental production of granulocyte colony-stimulating factor (G-CSF), in vivo, coincides with the onset of granulocytopoiesis in the developing fetus. To make this assessment, we obtained human placentae between 10 weeks of gestation and term and studied production of G-CSF in several ways. First, we sought to determine whether the onset of production of G-CSF mRNA in the placenta immediately precedes the appearance of neutrophil development in the fetus. Second, we assessed the effect of gestational age on the capacity of the placenta to generate G-CSF in vitro, by incubating cubes of placenta, with or without including interleukin-1 alpha (IL-1 alpha) in the culture media, and quantifying G-CSF in the cell culture supernatants 24 h later. Third, we assessed the rate of G-CSF production by the placenta, by perfusing two normal, term placentae using a membrane-oxygenator system, and quantifying G-CSF, at intervals, in the perfusates. We found: (1) no evidence that placental production of G-CSF is involved in regulating granulocytopoiesis in the fetus, (2) that the healthy placenta contains little or no G-CSF mRNA in vivo, (3) the placenta at term has a far greater capacity to produce G-CSF, when stimulated, than does the placenta before term, and (4) that although the placenta does not normally produce G-CSF in vivo, it has the capacity of generating very large quantities of G-CSF continuously over at least several days. (C) 1996 W. B. Saunders Company Ltd
We have cloned the cDNA encoding the human homologue of Smubp-2, which binds to single-stranded DNA with 5'-phosphorylated guanine-rich sequences related to the immunoglobulin mu chain switch (S(mu)) region. The deduced amino acid sequences of the mouse and human Smubp-2 are 76.5% homologous and contain motifs conserved among helicases. We have identified a domain essential for DNA binding at residues 638-786. The binding domain is less conserved (63% homologous) than the putative catalytic domain of N-terminal half containing most of the helicase motifs (85% homologous). The human and mouse SmuAbp-2 have similar, although slightly different, binding specificities. Although the mouse Smubp-2 preferentially binds to the mouse S(mu) motif (GGGGT), the human Smubp-2 binds equally well to the human (GGGCT) and mouse S(mu) motifs. The human Smubp-2 gene was mapped to chromosome 11 q13.2-q13.4 by in situ hybridization.
Selected regions of cloned EcoRI fragments of the chicken ovalbumin gene have been sequenced. The positions where the sequences coding for ovalbumin mRNA (ov-mRNA) are interrupted in the genome have been determined, and a previously unreported interruption in the DNA sequences coding for the 5' nontranslated region of the messenger has been discovered. Because directly repeated sequences are found at exon-intron boundaries, the nucleotide sequence alone cannot define unique excision-ligation points for the processing of a possible ov-mRNA precursor. However, the sequences in these boundary regions share common features; this leads to the proposal that there are, in fact, unique excision-ligation points common to all boundaries.
A new method for isolation of high molecular weight DNA from eukaryotes is presented. This procedure allows preparation of DNA from a variety of tissues such as calf thymus or human placenta and from cells which were more difficult to lyse until now (e.g. Crypthecodinium cuhnii, a dinoflagellate). The DNA obtained in such a way has an average molecular weight of about 200 X 10(6) d and contains very few, if any, single strand breaks.
The sequence of 72 base pairs of the rightward operator (O-R) of bacteriophage lambda is presented as determined with simple and rapid methods for direct DNA sequencing. The sequence of an operator mutant is also described. The methods are of general use in sequencing DNA fragments with unique 5' ends up to 50 base pairs in length. Previous experiments have shown that this operator contains multiple sites recognized by the lambda phage repressor. We believe we have identified three of these sites.
We present the sequence of regions of the chicken ovalbumin gene believed to be important in the control of initiation of
transcription, splicing, and transcription termination or polyadenylation. Comparison with corresponding areas of other genes
reveals some homologous regions which might play a role in these processes.
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.
Cultures of a rat myogenic cell line were used to examine the question of whether in proliferating precursor cells genes which
are programmed to be expressed later in development, in the same cell lineage, differ in DNAase I sensitivity from genes which
are never expressed in these cells. Nuclei isolated from proliferating mononucleated myoblasts, differentiated cultures containing
multinucleaged fibers, and rat brain, were treated with DNAase I. The sensitivity of the genes coding for the muscle-specific
α-actin, myosin light chain 2 and the nonmuscle β-actin was measured by blot hybridization of nuclear DNA with the corresponding
cloned cDNA and genomic DNA probes. The sensitivity of these genes was compared to that of a gene not expressed in the muscle
tissue. The results showed that in the muscle precursor cells, the potentiality of tissue-specific genes to be expressed is
not reflected in DNAase I sensitivity. The changes which render these genes preferentially sensitive to DNAase I take place
during the transition to terminal differentiation. The results showed also that the region of DNAase I sensitivity of the
α-actin gene in the differentiated cells ends between 40 to 700 bp 5' to the structural gene. No DNAase I hypersensitive site
was detected 5' to the αactin gene.
We report the complete nucleotide sequence of a human beta actin cDNA. Both the 5' and 3' untranslated regions of the sequence are similar (greater than 80%) to the analogous regions of the rat beta-actin gene reported by Nudel et al (1983). When a segment of the 3' untranslated region is used as a radiolabelled probe, strong hybridization to chick beta actin mRNA is seen. This conservation of sequences suggests that strong selective pressures operate on non-translated segments of beta actin mRNA.
A recombinant phage containing an actin gene (lambda Ha201) was isolated from a human DNA library and the structure of the actin gene was determined. The amino acid sequences deduced from the nucleotide sequences of lambda Ha201 were compared with those of six actin isoforms; they matched those of bovine aortic smooth muscle actin, except for codon 309, which was valine (GTC) in lambda Ha201 and alanine (GCN) in bovine aortic smooth muscle actin. Southern blot hybridization experiments showed that the gene of normal human cells did not have the TaqI-sensitive site around position 309, whereas half of the genes of HUT14 cells did. These results indicate that one allele of the aortic smooth muscle actin gene in HUT14 cells has a transition point mutation (C----T) at codon 309 and that the amino acid sequences of normal human aorta and bovine smooth muscle actins are probably identical. In addition to the five introns interrupting exons at codons 150, 204, and 267, and between codons 41 and 42 and 327 and 328, which are common to skeletal muscle and cardiac muscle actin genes, the smooth muscle actin gene has two more intron sites between codons 84 and 85 and 121 and 122. The previously unreported intron site between codons 84 and 85 is unique to the smooth muscle actin gene. The intron site between codons 121 and 122 is common to beta-actin genes but is not found in other muscle actin genes. A hypothesis is proposed for the evolutionary pathway of the actin gene family.
Two 19-base-long oligonucleotides were synthesized, one complementary to the normal human beta-globin gene (beta A) and one complementary to the sickle cell beta-globin gene (beta S). The nonadecanucleotides were radioactively labeled and used as probes in DNA hybridization. Under appropriate hybridization conditions, these probes can be used to distinguish the beta A gene from the beta S allele. The DNA from individuals homozygous for the normal beta-globin gene (beta A beta A) only hybridized with the beta A specific probe; the DNA from those homozygous for the sickle cell beta-globin gene (beta S beta S) only hybridized with the beta S specific probe. The DNA from heterozygous individuals (beta A beta S) hybridized with both probes. This allele-specific hybridization behavior of oligonucleotides provides a general method for diagnosis of any genetic disease which involves a point mutation in the DNA sequence of a single-copy gene.
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.
cDNA clones encoding three classes of human actins have been isolated and characterized. The first two classes (gamma and beta, cytoplasmic actins) were obtained from a cDNA library constructed from simian virus 40-transformed human fibroblast mRNA, and the third class (alpha, muscle actin) was obtained from a cDNA library constructed from adult human muscle mRNA. A new approach was developed to enrich for full-length cDNAs. The human fibroblast cDNA plasmid library was linearized with restriction enzymes that did not cut the inserts of interest; it was then size-fractionated on gels, and the chimeric molecules of optimal length were selected for retransformation of bacteria. When the resulting clones were screened for actin-coding sequences it was found that some full-length cDNAs were enriched as much as 50- to 100-fold relative to the original frequency of full-length clones in the total library. Two types of clones were distinguished. One of these clones encodes gamma actin and contains 100 base pairs of 5' untranslated region, the entire protein coding region, and the 3' untranslated region. The second class encodes beta actin, and the longest such clone contains 45 base pairs of 5' untranslated region plus the remainder of the mRNA extending to the polyadenylic acid tail. A third class, obtained from the human muscle cDNA library, encodes alpha actin and contains 100 base pairs of 5' untranslated region, the entire coding region, and the 3' untranslated region. Analysis of the DNA sequences of the 5' end of the clones demonstrated that although beta- and gamma-actin genes start with a methionine codon (MET-Asp-Asp-Asp and MET-Glu-Glu-Glu, respectively), the alpha-actin gene starts with a methionine codon followed by a cysteine codon (MET-CYS-Asp-Glu-Asp-Glu). Since no known actin proteins start with a cysteine, it is likely that post-translational removal of cysteine in addition to methionine accompanies alpha-actin synthesis but not beta- and gamma-actin synthesis. This observation has interesting implications both for actin function and actin gene regulation and evolution.
Malignant human fibroblasts, transformed in vitro by a single chemical treatment, and the untransformed parental cells have been compared by two-dimensional gel electrophoresis of their proteins. One transformed cell line, HUT-14, exhibits an abundance of a new polypeptide, A' (pI 5.2; molecular weight 44,000), amounting to approximately 3% of the cellular protein. There are at least 23 additional differences in polypeptides out of greater than 1000 electrophoretically distinguishable species. The A' polypeptide has been identified as a variant form of actin by immunoprecipitation with anti-actin antibody and comparison of its tryptic peptide patterns with those produced by beta- and gamma-actin polypeptides also found in HUT-14 cells. A' is distinguishable from the normal forms of actin (alpha, beta, and gamma polypeptides) in that it is more acidic and migrates at a slower rate in sodium dodecyl sulfate gels. Synthesis of A' may occur as a result of a somatic mutation affecting one of the normal actin genes. The electrophoretic behavior of A' in two-dimensional gels is compatible with several mutation models.
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.
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.
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.
Cloned beta-globin genes of both mouse and rabbit each contain a large and a small intervening sequence (intron) of about equal length at precisely the same positions relative to the coding sequence. The homologous introns show some sequence similarity, particularly at the junctions with the coding sequence. They most probably arose from a common ancestral sequence and diverged substantially during evolution.
DNA can be sequenced by a chemical procedure that breaks a terminally labeled DNA molecule partially at each repetition of a base. The lengths of the labeled fragments then identify the positions of that base. We describe reactions that cleave DNA preferentially at guanines, at adenines, at cytosines and thymines equally, and at cytosines alone. When the products of these four reactions are resolved by size, by electrophoresis on a polyacrylamide gel, the DNA sequence can be read from the pattern of radioactive bands. The technique will permit sequencing of at least 100 bases from the point of labeling.
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.
Cultured fibroblast cells derived from a skin biopsy sample taken from normal human adult were exposed to a potent carcinogen, 4-nitroquinoline 1-oxide. Alterations of cell growth pattern such as higher density and piling up of cells were noticed in some fractions of cultures that were successively subcultured after nitroquinoline oxide treatment. Morphologically altered cells retained this growth pattern and became established lines of transformed cells without showing the limited life-span characteristic of normal cells in culture. The transformed cells showed a higher saturation density and the ability to grow in soft agar, properties that are usually correlated with neoplastic transformation of cells in culture. Selection of preexisting transformed human cells as a mechanism of this observed transformation seemed unlikely because clones of these normal cells could also be used to assess the transforming effect of nitroquinoline oxide. Preliminary results suggest that numerous cell divisions were required for the development of the transformation after nitroquinoline oxide treatment of these human cells.
When the transformed cell lines were injected subcutaneously into nude (athymic) mice, solid tumors were produced at the site of inoculation.
Treatment with N-methyl-N′-nitro-N-nitrosoguanidine also induced cell transformation, in a manner similar to treatment with nitroquinoline oxide. However, transformation was not induced with (i) 4-aminoquinoline 1-oxide (a noncarcinogenic derivative of 4-nitroquinoline 1-oxide), (ii) 3-methylcholanthrene (a carcinogen that cannot be metabolically activated by the target cells employed), or (iii) the solvent dimethyl sulfoxide.
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.
During the late stage of adenovirus 2 infection, RNA chains are initiated at a site near coordinate 16 (Evans et al., 1977) and transcribed ∼30,000 nucleotides to the far end of the genome at coordinate 100. Late mRNAs processed from these transcripts contain a common spliced tripartite leader (Berget, Moore and Sharp, 1977; Chow et al., 1977a) encoded at ∼16, 20 and 27, and protein coding sequences which map down-stream. This report maps the late promoter and the capped 5′ end of nuclear and cytoplasmic RNAs from this transcription unit, and analyzes their structures. We show that nascent RNA chains pulse-labeled in vivo are initiated at coordinate 16.5 ± 0.5 and contain the sequences intervening between the leader segments. We map the capped 5′ terminus of late nuclear transcripts at a site between 16.4 and 16.6 by aligning T1 RNAase oligonucleotides from nuclear RNA with the DNA sequence of the promoter region. The structure of the first eleven residues of the capped 5′ terminus of late mRNA was determined by direct RNA sequencing. This structure corresponds exactly to a DNA sequence at coordinate 16.4 and precisely positions the mRNA cap template within the promoter region. These results suggest that the promoter and the cap template sites are coincident, and that the initiating residues of the primary transcript are precursors of the capped 5′ end of mRNA.
The sequence A-A-U-A-A-A is present in six different purified messenger RNA molecules (specifically the alpha-and beta-globulin mRNAs of rabbit and human, the immunoglobulin light chain mRNA of mouse (MOPC 21) and the ovalbumin mRNA of chicken) about 20 residues away from the 3'-terminal poly (A) sequence. In addition, a large selection of the 3' non-coding regions of rabbit and human globulin mRNAs (both the alpha and beta globin mRNAs) are 85% homologous, demonstrating that this region is significantly conserved in evolution.
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.
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.
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.
The nucleotide sequence of the rat beta-actin gene was determined. The gene codes for a protein identical to the bovine beta-actin. It has a large intron in the 5' untranslated region 6 nucleotides upstream from the initiator ATG, and 4 introns in 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 beta-actin 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 in the beta-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.
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.
With the development of large data banks of protein and nucleic acid sequences, the need for efficient methods of searching such banks for sequences similar to a given sequence has become evident. We present an algorithm for the global comparison of sequences based on matching k-tuples of sequence elements for a fixed k. The method results in substantial reduction in the time required to search a data bank when compared with prior techniques of similarity analysis, with minimal loss in sensitivity. The algorithm has also been adapted, in a separate implementation, to produce rigorous sequence alignments. Currently, using the DEC KL-10 system, we can compare all sequences in the entire Protein Data Bank of the National Biomedical Research Foundation with a 350-residue query sequence in less than 3 min and carry out a similar analysis with a 500-base query sequence against all eukaryotic sequences in the Los Alamos Nucleic Acid Data Base in less than 2 min.
From a human gene library we have isolated and sequenced a beta-actin-like pseudogene, H beta Ac-psi 2, which lacks intervening sequences and contains several mutations resulting in frame-shifts, stop codons and in a departure from the known beta-actin protein sequence. We have also extended our sequence work on the intronless human beta-actin-related pseudogene H beta Ac-psi 1 described previously and we find that both genes are processed genes ending in a poly(dA) tract and flanked by direct repeats. The gene H beta Ac-psi 2 is preceded by a 230-bp region in which the simple sequence 5'-GAAA-3' is repeated greater than 40 times. This satellite-like sequence is highly repetitive in the human genome.
We report here a comparison of the nucleotide sequences of the chick and rat skeletal muscle alpha-actin genes which reveals strongly conserved blocks of sequence in the putative transcriptional promoter and 3'-untranslated regions. This is the first instance of strong sequence conservation in untranslated gene regions between birds and mammals, other than the CAAT and ATA promoter homologies and the putative poly(A) addition signal, AATAAA. The strong conservation of these sequences suggests that they may have a role in the tissue-specific expression of the skeletal muscle alpha-actin gene.
HUT 14 is a cloned transformed cell line derived from normal diploid human KD fibroblasts. HUT 14 cells have an altered actin phenotype. In addition to the two nonmuscle actins beta and gamma, also present in the parent KD cells, they show the stable expression of a novel actin species (Ax-actin). Amino acid sequence analysis has been used to identify the three actins of HUT 14 cells. beta- and gamma-actins are identified as normal mammalian nonmuscle actins whereas Ax-actin is characterized as a beta-actin mutant revealing a single amino acid substitution at position 244. The results obtained are compatible with a simple mutational event involving a point mutation in one of the two beta-nonmuscle actin genes assumed to be present in proliferating human diploid fibroblasts. Certain emerging principles of nonmuscle actin gene expression in higher vertebrates are discussed.
The expression of RNA sequences coding for myofibrillar proteins has been followed during terminal differentiation in a mouse skeletal muscle cell line. Cloned complementary DNA probes hybridizing with the actins, skeletal muscle α-actin, myosin heavy chain and the myosin alkali light chains were employed in Northern blotting experiments with total cellular poly (A)-containing RNA extracted from the cultures at different times after plating. At the same times, parallel cultures were pulse-labelled with [35S]methionine and the pattern of newly synthesized proteins was analysed by two-dimensional gel electrophoresis. Synthesis of skeletal muscle α-actin and of the myosin alkali light chains (LClemb, LC1, LC3) was not detectable in dividing myoblast cultures. From the onset of cell fusion, the synthesis of myosin heavy chain, LClemb and α-actin increases with similar kinetics. Synthesis of LC3 (and trace amounts of LC1F) is detectable and subsequently increases at later stages of myotube formation. The corresponding messenger RNAs coding for myosin heavy chain and skeletal muscle α-actin are first detectable immediately before the initiation of myofibrillar protein synthesis. mRNAs coding for the non-muscle actins are accumulated in myoblasts and diminish after cell fusion. Comparisons between muscle mRNAs depend on the relative sensitivities of the different probes, reflecting mainly their homology with the isoform of the actin or myosin multigene family expressed. Quantitative analysis of Northern blots gives an estimated increase in skeletal muscle α-actin mRNA, with an homologous probe, of at least 130-fold with a minimum level of detection of 40 to 80 molecules per cell. Accumulation of this species and of the myosin heavy chain mRNA follows similar kinetics. mRNA coding for LC3, the principal myosin light chain detected with the probe, appears to accumulate to a lesser extent initially, paralleling synthesis of the corresponding protein. These results using cloned probes demonstrate a close temporal correlation between muscle mRNA accumulation and protein synthesis during terminal myogenesis in this muscle line.
Comparison of about 50 pairs of homologous nucleotide sequences for different genes revealed that the substitutions between synonymous codons occurred at much higher rates than did amino acid substitutions. Furthermore, five pairs of mRNA sequences for different genes were compared in species that had diverged at the same time. The evolutionary rate of synonymous substitution was estimated to be 5.1 X 10(-9) per site per year on the average and is approximately constant among different genes. It also is suggested that this property would be suitable for a molecular clock to determine the evolutionary relationships and branching order of duplicated genes. Each functional block of the noncoding region evolves with a rate that is almost constant, regardless of the types of genes. The intervening sequence and the 5' portion of the 3' noncoding region show considerable divergence, the extent of which is almost comparable to that in the synonymous codon sites, whereas the other blocks consisting of the 5' noncoding region and the 3' portion of the 3' noncoding region are strongly conserved, showing approximatley half of the divergence of the synonymous sites. This strong sequence preservation might be due to the functional requirements for transcription and modification of mRNA.
By in vitro translation, we have identified the mRNA species that codes for a novel actin polypeptide (Ax-actin) in the chemically transformed human fibroblast line HuT-14. The relatedness of the coding sequences of the Ax- and beta-actin genes is indicated by our finding that pcDd actin ITL-I DNA, a recombinant plasmid DNA that contains a DNA sequence complementary to actin mRNA of Dictyostelium discoideum, hybridizes both the Ax-actin mRNA and the beta-actin mRNA but not the gamma-actin mRNA. In contrast, pcHa-1 DNA, a recombinant plasmid constructed by cloning a DNA sequence complementary to human actin mRNA from HuT-14 cells into pBR322, hybridized to all three mRNA species. In addition, no difference was observed between Ax- and beta-actin mRNAs when their molecular size was determined either by sucrose density gradient sedimentation or by methyl mercury agarose gel electrophoresis. Southern blot transfer of radioactive pcDd actin DNA to restriction endonuclease-digested Hut-14 DNA produced only a single hybrid band (a 6-kilobase fragment); the pcHa-1 DNA probe detected one additional band (a 3-kilobase fragment). These results suggest that HuT-14 cells contain only one copy per haploid genome for Ax- or beta-actin. When considered together with recent determination of the entire amino acid sequences of Ax- and beta-actin, our findings indicate that Ax-actin is the product of a mutated beta-actin gene and are evidence for the occurrence of a mutation in a chemically transformed cell.
We present the results of a detailed comparison of the primary structure of human beta-like globin genes and their flanking sequences. Among the sequences located 5' to these genes are two highly conserved regions which include the sequences ATA and CCAAT located 31 +/- 1 and 77 +/- 10 bp, respectively, 5' to the mRNA capping site. Similar sequences are found in the corresponding locations in most other eucaryotic structural genes. Calculation of the divergence times of individual beta-like globin gene pairs provides the first description of the evolutionary relationships within a gene family based entirely on direct nucleotide sequence comparisons. In addition, the evolutionary relationship of the embryonic epsilon-globin gene to the other human beta-like globin genes is defined for the first time. Finally, we describe a model for the involvement of short direct repeat sequences in the generation of deletions in the noncoding and coding regions of beta-like globin genes during evolution.
The complete nucleotide sequence of the actin gene from Saccharomyces cerevisiae has been determined. The coding region is interrupted by a 304-base-pair intervening sequence that is located within the triplet coding for amino acid 4. DNA sequences of the intron-exon junctions are similar to those found in higher eukaryotes and can be aligned such that the intron starts with the dinucleotide 5'-G-T-3' and ends with 5'-A-G-3'. Regions fo homology within the sequences upstream from the initiation codon and those following the termination codon have been detected between the yeast iso-1-cytochrome c gene and the actin gene. As deduced from the nucleotide sequence, yeast actin has 374 amino acid residues. Its primary structure, especially the NH2-terminal third of the protein, is highly conserved during evolution.
The yeast Saccharomyces cerevisiae is known to contain the highly conserved and unbiquitous protein actin. We have used cloned actin sequences from Dictyostelium discoideum to identify and clone the actin gene in yeast. Hybridization to genomic fragments of yeast DNA suggest that there is a single actin gene in yeast. We have determined the nucleotide sequence of that gene and its flanking regions. The sequence of the gene reveals an intervening sequence of 309 base pairs in the coding sequences at the 5' end of the gene. The existence and location of the intervening sequence was verified by using the dideoxy chain termination technique to determine the sequence at the 5' terminus of the actin mRNA. The similarity of the splice junction sequences in this gene to those found in higher eukaryotes suggests that yeast must possess a similar splicing enzyme.
He have determined the complete nucleotide sequence of a sea urchin actin gene, including the entire protein-coding sequence,
introns and approximately 500 and 700 nucleotides adjacent to protein-coding-sequence on the 5′ and 3′ sides, respectively.
This gene is split between codons 121 and 122 and within codon 204 by two introns which are 233 and 181 nucleotides in length,
respectively. Comparison of the sequence of the two introns indicates a region of distant relatedness which covers about 25%
of their lengths, suggesting that these sequences might have derived from a common ancestral sequence. The encoded amino acid
sequence, which matches closely with that reported for actins from other species, is more cytoplasmic-like than muscle-like
when compared to vertebrate actins. Analysis of the coding-flanking regions indicates the presence of sequences similar to
those thought to be important for initiation of transcription and poly-adenylation of mRNA. The location of these sequences
and the size of an actin mRNA, transcribed from this or a very closely related gene, suggests that initiation occurs 347 nucleotides
51 of coding and polyadenylation approximately 515 nucleotides 3′ of coding.
Treatment of cultured human diploid fibroblasts with a chemical carcinogen produced a clonal neoplastic cell line (HUT-14) that expresses a mutant β actin, nearly an equal amount of normal β actin and one additional nonmuscle actin species, γ actin. These three actins are the principal structural components of the detergent-resistant cytoskeleton. A substrain of HUT-14 was derived from a tumor produced by inoculation of a nude mouse with a highly selected subclone of HUT-14 cells. Cells of this new substrain, HUT-14T, exhibit a more variant distribution of cytoskeletal actin than the parent HUT-14 strain and a further diminution in cytoskeletal fibronectin. HUT-14T is also elevated in tumorigenicity, producing larger, faster-growing fibrosarcomas in the nude mouse than the parent HUT-14 strain with fewer inoculated cells. These phenotypic cellular changes accompany a biochemical and functional change in the mutant β-actin polypeptide. The more variant mutant actin of HUT-14T differs from the original mutant polypeptide by: one additional negative net charge, a short half-life in the cell, a greatly diminished ability to incorporate into the detergent-resistant cytoskeleton, a decrease in affinity for deoxyribonuclease I and a faster rate of synthesis. It appears that the mutant actin of HUT-14 acquired a second-site mutation that was selected during a subcloning step prior to derivation of the HUT-14T substrain. The hypothesis of a second-site mutation is supported by the finding that the new β-actin species in HUT-14T cells is translated correctly from HUT-14T mRNA in vitro. The increased rate of synthesis of mutant β actin in HUT-14T cells is accompanied by an approximate doubling in the relative amount of translatable mutant β-actin mRNA, an event that occurred separately from the event that produced the altered mutant β actin. These separate variations in β-actin expression are accompanied by incremental increases of malignant potential in this cell line.
We described the construction of an α-actin complementary deoxyribonucleic acid (cDNA) clone, pAC269 [Schwartz, R. J., Haron, J. A., Rothblum, K. N., & Dugaiczyk, A. (1980) Biochemistry 19, 5883], that was used as a hybridization probe in the current investigation to examine the induction of actin messenger ribonucleic acid (mRNA) during myogenesis. A Tm difference of 10-13°C between skeletal muscle a-actin and nonmuscle β- and γ-actin mRNAs and pAC269 allowed us to establish the highly stringent hybridization conditions necessary to measure separately the content of α-actin mRNA and β- and γ-actin mRNA during muscle development in culture. We observed low levels of α-actin mRNA (∼130 molecules/cell) in replicating profusion myoblasts. The vast majority of actin mRNA (2000 molecules/cell) present at this stage was accounted for by β- and γ-actin mRNA. Beginning at myoblast fusion, α-actin mRNA accumulated and within 30 h reached a level 270-fold greater than that observed in the undifferentiated state. At 95 h in culture when myotube formation was completed, α-actin content was at its peak (36 000 molecules/nucleus). Conversely, β- and γ-actin mRNA content began to decline at the beginning of fusion, and by the end of myotube formation β-and γ-actin mRNAs were undetectable by our techniques. A rapid depression of α-actin mRNA levels was observed after 95 h in the absence of cell death. At 6 days after the initiation of myotube formation, the content of α-actin mRNA was reduced by 80% in comparison to peak values and remained at that level. The switching of actin mRNA species was inhibited in myoblasts treated with bdU. The accumulation of α-actin mRNA and the disappearance of β- and γ-actin mRNA were observed following the reversal of the bdU block and coincident with the onset of myoblast fusion. We found that the expression of actin genes within the actin multigene family is switched in myogenesis through a strict developmental pattern.
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