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L Kedes

Publications and source records attributed to L Kedes.

At least 91 records · Page 5Linked to original sources
PubMed1989

Regulation of the human beta-actin promoter by upstream and intron domains.

We have identified three regulatory domains of the complex human beta-actin gene promoter. They span a region of about 3000 bases, from not more than -2011 bases upstream of the mRNA cap site to within the 5' intron (832 bases long). A distal upstream domain contains at least one enhancer-like element. A proximal upstream domain, with a CArG [for CC(A + T rich)6GG] motif found in all known mammalian actin genes, seems to confer serum, but not growth factor, inducibility. The third domain is within the evolutionarily conserved 3' region of the first intron and contains a 13 base-pair sequence, identical to the upstream sequence with the CArG motif. This domain also contains sequences that are both serum and fibroblast growth factor inducible.

Actins↗
PubMed1989

Identification and characterization of a factor that binds to two human sarcomeric actin promoters.

A gel mobility shift assay was used to examine nuclear extracts of muscle and nonmuscle cell lines for a factor or factors that interact with the functional promoter segment of both the human cardiac and skeletal alpha-actin genes. A single major band of altered mobility was seen with nuclear extracts from C2 myoblasts and myotubes and L8 myoblasts and myotubes as well as several non-muscle cell lines. Competition experiments with nuclear extracts from both muscle and non-muscle cells localized the region of binding to a 10-base pair sequence of the cardiac alpha-actin gene containing the functional CArG box promoter domain. Similar results and binding site specificities were obtained with a DNA fragment from the human skeletal alpha-actin promoter. The CArG box binding factor was shown to be distinct from a CAAT box binding factor. A molecular weight of 67 kDa for the CArG box binding factor was determined by photoaffinity labeling of the complex and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protease treatment resulted in a smaller fragment that still bound the cardiac alpha-actin promoter fragment and may represent a structurally distinct DNA-binding domain. We discuss how muscle-specific expression of the sarcomeric alpha-actin genes might be achieved with a ubiquitous transcription factor.

Actins↗
PubMed1989

DNA bending is induced by a transcription factor that interacts with the human c-FOS and alpha-actin promoters.

Conserved sequence elements in the human cardiac and skeletal alpha-actin promoters that contain the CC(A + T-rich)6GG motif have been shown to regulate transcription of these genes. A similar sequence is found in the serum response element of the human c-FOS gene. In this study, we demonstrate that indistinguishable proteins bind to each of five CC(A + T-rich)6GG elements examined in the human cardiac and skeletal alpha-actin promoters and the c-FOS serum response element. Using electrophoretic techniques, we show that these factors induce a stable bend in the DNA upon binding, and the bend center is shown to coincide with the CC(A + T-rich)6GG element. In addition, the ability to bend DNA is retained by a small proteolytic fragment of the protein, suggesting that the DNA-binding domain of the protein is resistant to proteases and is sufficient to bend DNA.

Actins↗
PubMed1989

The sarcomeric actin CArG-binding factor is indistinguishable from the c-fos serum response factor.

The c-fos serum response element (SRE) and a sarcomeric actin promoter element (CArG box) are similar in sequence and are recognized, respectively, by the serum response factor (SRF) and the CArG-binding factor (CBF). Although the transcriptional controls for the c-fos and sarcomeric actin genes are rather different, SRF and CBF have been found to be indistinguishable by all criteria tested. They exhibited similar chromatographic properties, sedimentation rates, and temperature stabilities. In mobility shift assays, the SRE competed more strongly than the actin CArG box for formation of either the SRF-SRE or the CBF-CArG complex. The symmetric inverted repeat of the left side of the Xenopus cytoskeletal actin SRE also competed, even more strongly, for each complex. The site-specific binding of each protein was inhibited both by orthophenanthroline, whose effects were reversed by zinc addition, and by treatment with potato acid phosphatase. Furthermore, immune serum raised against the c-fos SRF also recognized the actin CBF. We discuss how transcriptional control of these diverse genes might be obtained with a single similar factor.

Animals↗
PubMed1989

Identification of multiple proteins that interact with functional regions of the human cardiac alpha-actin promoter.

5' Sequences of the human cardiac alpha-actin gene are involved in the tissue-specific and developmental regulation of the gene. Deletion analyses combined with transient expression experiments in muscle cells have demonstrated three primary regions of functional importance (A. Minty and L. Kedes, Mol. Cell. Biol. 6:2125-2136, 1986; T. Miwa and L. Kedes, Mol. Cell. Biol. 7:2803-2813, 1987), and we have previously demonstrated binding of a protein indistinguishable from serum response factor (SRF) to the most proximal region (T.A. Gustafson, T. Miwa, L.M. Boxer, and L. Kedes, Mol. Cell. Biol. 8:4110-4119, 1988). In this report, we examine protein interaction with the remainder of the promoter. Gel shift and footprinting assays revealed that at least seven distinct nuclear proteins interacted with known and putative regulatory regions of the promoter. The transcription factor Sp1 bound to eight sites, as demonstrated by footprinting assays and gel shift analysis with purified Sp1. Purified CCAAT box-binding transcription factor CTF/NF-I and Sp1 were shown to interact with the far-upstream regulatory element at -410, and footprint analysis showed extensive overlap of these two sites. Two unidentified proteins with similar but distinct footprints interacted with the second region of functional importance at -140, which contains the second CArG motif [CC(A + T rich)6GG], and these proteins were shown to be distinct from SRF. SRF was found to bind to the remaining three CArG boxes, two of which were closely interdigitated with Sp1 sites. In addition, CArG box 4 was found to interact with SRF and another distinct protein whose footprint was contained within the SRF-binding site. Sequences surrounding the TATA box were also shown to bind proteins. Sp1 was shown to bind to a site immediately downstream from the TATA box and to a site within the first exon. Thus, each of the three functional upstream regions, as defined by transfection assays, was shown to interact with five factors: Sp1 and CTF/NF-I at the upstream site, two unidentified proteins at the central site, and SRF at the most proximal site. These results suggest that expression of the cardiac actin gene in muscle cells is controlled by complex interactions among multiple upstream and intragenic elements.

Actins↗
PubMed1988

Differential expression of slow and fast skeletal muscle troponin C. Slow skeletal muscle troponin C is expressed in human fibroblasts.

We have isolated and sequenced the cDNAs for human slow and fast skeletal muscle troponin C (TnC). Each cDNA is encoded by one of the two TnC genes in the human genome. The fast skeletal muscle TnC gene appears to be expressed exclusively in skeletal muscle. Only the slow TnC gene is expressed in human cardiac ventricle. The slow skeletal TnC gene is also expressed in skeletal muscle and, surprisingly, in several human fibroblast cell lines. Thus, at least one of the three proteins of the troponin complex appears to be expressed in non-muscle cells of higher vertebrates. The relative steady-state amounts of the slow and fast skeletal TnC mRNAs in various adult and embryonic striated muscles are similar to the expected amounts of the corresponding protein, suggesting that the expression of TnC genes is controlled predominantly by the production or accumulation of mRNA rather than by translational or post-translational mechanisms.

Amino Acid Sequence↗
PubMed1988

Adenovirus E1A products suppress myogenic differentiation and inhibit transcription from muscle-specific promoters.

The primary function of the adenovirus E1A-region genes is to activate other adenoviral genes during a permissive viral infection by modifying the host cell transcriptional apparatus. Host cell immortalization, or transformation by the whole adenoviral early region, presumably results as a consequence of these modifications. Both transcriptional activation and transcriptional repression of non-adenoviral genes by the E1A proteins have been reported. It is currently not clear which, if either, of these activities contributes to host cell transformation and immortalization. Although there may be a physiological impact of some E1A-stimulated host cell genes, in many cases the functional significance is unclear. No common target sequences have been recognized in stimulated cellular genes and it has recently been proposed that in many cases, particularly involving newly transfected genes, available 'TATA-box' sequences may be the opportunistic beneficiaries of E1A assistance as a secondary consequence of E1A primary functions within the host cell nucleus. E1A-mediated transcriptional repression appears to be a more specific process insofar as common core elements are shared by the E1A-suppressed SV40, polyoma B, IgG heavy-chain and insulin enhancers. In the present communication we report that the complete myogenic programme of L8 and C2 myoblasts can be blocked by the introduction of constitutively expressing E1A genes, and show that the transcriptional induction of muscle-specific genes is inhibited. In particular, the promoter-inducing activities of well-defined elements that are required for the muscle-specific expression of the two sarcomeric alpha-actins, and which normally bind cellular trans-acting factors, become targets for E1A suppression. The results support the hypothesis that the suppression of differentiation by E1A products is effected by an E1A-mediated block in the transcriptional activation of cellular genes by specific developmentally regulated cis-acting promoter elements.

Adenovirus Early Proteins↗
PubMed1988

Nucleotide sequence and expression of the human skeletal alpha-actin gene: evolution of functional regulatory domains.

Regulation of the actin multigene family involves the recognition of regulatory sequences that specify the tissue type and developmental program of expression for each actin isotype. In order to investigate the underlying regulatory mechanisms, the human skeletal alpha-actin gene and its 5' regulatory region have been cloned and sequenced. This actin gene has seven exons; there is one large intron in the 5' untranslated region which is characteristic of the actins and many muscle-specific genes. The 5' flanking sequences are sufficient to direct tissue-specific and differentiation-regulated expression when transfected into the heterologous rat L8 myogenic cells, indicating a highly conserved regulatory system. The DNA sequence was compared to that of other actin genes, and several regions of sequence similarity were identified, particularly within regions known to be important for gene expression. Most notable among the conserved sequences are the CC(A/T rich)6GG (CArG box) motifs which have demonstrated interactions with trans-acting transcriptional factors. This same motif has been identified in several other genes and in some also serves as a binding site for transcription regulatory factors.

Actins↗
PubMed1988

In vivo system for characterizing clonal variation and tissue-specific gene regulatory factors based on function.

The inducibility of stably transfected alpha-cardiac actin genes differs among L cell clones. We examined the ability of muscle-specific factors to induce the expression of the human muscle alpha-cardiac actin gene promoter when stably transfected into mouse fibroblast L cells. This promoter is transcriptionally active in L cells at a low level, 2-5% of that in transfected muscle cells. Upon fusion with muscle cells to form heterokaryons, expression of the transfected alpha-cardiac actin gene promoter can be induced. However, induction is observed with only 10% of transfected L cell clones and the magnitude of this induction varies between 5- and 50-fold. These properties of the transfected L cell appear to be stably inherited. Our results are consistent with the hypothesis that muscle cells contain factors capable of increasing the transcription of the transfected gene, but that differences among L cell clones, possibly in the site of integration in the genome, determine the extent to which the gene can respond. By fusion into heterokaryons, transfectants with responsive genes can be identified. Such clones should prove useful in determining the basis for clonal variation. In addition, they provide an in vivo system for isolating functionally active tissue-specific transcription factors and the genes that encode them.

Acetyltransferases↗
PubMed1988

Interaction of nuclear proteins with muscle-specific regulatory sequences of the human cardiac alpha-actin promoter.

The human cardiac alpha-actin promoter is involved in the muscle-specific transcriptional regulation of the gene. In this study, we utilized gel mobility shift, methylation interference, and DNase I protection assays to examine protein factor interaction with the promoter in vitro. All assays demonstrated specific interaction of nuclear factors with a region of the promoter encompassed by nucleotides -93 to -113 base pairs from the transcriptional start site. This region contains a CC(A + T-rich)6GG element, termed a CArG box, which has previously been implicated in the muscle-specific transcriptional regulation of the gene by functional assays. Although the gene is only expressed in muscle cells, identical binding activity was present in nuclear extracts of all cell types examined, including those of muscle (C2, L8, and L6 cells) and nonmuscle (HeLa, NIH 3T3, HuT12, and L cells) origin. Furthermore, methylation interference assays showed that identical nucleotides interacted with factors isolated from C2 and HeLa cells. Competition studies showed that the CArG-binding factor, designated as CBF, also interacts with the c-fos serum responsive element, which contains a CArG element, but not with the simian virus 40 enhancer and early promoter. Thus, a region of the human cardiac alpha-actin promoter known to be functionally involved in muscle-specific regulation of the gene appears to interact in vitro, and in an identical manner, with a factor(s) which is neither muscle nor gene specific, suggesting a more complex mode of regulation than previously envisioned.

Actins↗
PubMed1988

A common factor regulates skeletal and cardiac alpha-actin gene transcription in muscle.

The skeletal and cardiac alpha-actin genes are coexpressed in muscle development but exhibit distinctive tissue-specific patterns of expression. We used an in vivo competition assay and an in vitro electrophoretic mobility shift assay to demonstrate that both genes interact with a common trans-acting factor(s). However, there was at least one gene-specific cis-acting sequence in the skeletal alpha-actin gene that interacted with a trans-acting factor which was not rate limiting in the expression of the cardiac alpha-actin gene. The common factor(s) interacted with several cis-acting regions that corresponded to sequences that are required for the transcriptional modulation of these sarcomeric alpha-actin genes in muscle cells. These regulatory regions contained the sequence motif CC(A + T-rich)6GG, which is known as a CArG box. Results of in vivo competition assays demonstrated that the factor(s) bound by the skeletal alpha-actin gene is also essential for the maximal activity of the cardiac alpha-actin, simian virus 40 (SV40), alpha 2(I)-collagen, and the beta-actin promoters in muscle cells. In contrast, fibroblastic cells contained functionally distinct transcription factor(s) that were used by the SV40 enhancer but that did not interact with the sarcomeric alpha-actin cis-acting sequences. The existence of functionally different factors in these cell types may explain the myogenic specificity of these sarcomeric alpha-actin genes. Results of in vitro studies suggested that both the sarcomeric alpha-actin genes interact with the CArG box-binding factor CBF and that the skeletal alpha-actin promoter contains multiple CBF-binding sites. In contrast, CBF did not interact in vitro with a classical CAAT box, the SV40 enhancer, or a linker scanner mutation of an alpha-actin CArG box. Furthermore, methylation interference and DNase I footprinting assays demonstrated the precise sites of interaction of CBF with three CArG motifs at positions -98, -179, and -225 in the human skeletal alpha-actin gene.

Actins↗
PubMed1988

Structure, chromosome location, and expression of the human gamma-actin gene: differential evolution, location, and expression of the cytoskeletal beta- and gamma-actin genes.

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.

Actins↗
PubMed1988

Modulation of microfilament protein composition by transfected cytoskeletal actin genes.

HuT-14T is a highly tumorigenic fibroblast cell line which exhibits a reduced steady-state level of beta-actin due to coding mutations in one of two beta-actin alleles. The normal rate of total actin synthesis could be restored in some clones of cells following transfection of the functional beta-actin gene but not following transfection of the functional gamma-actin gene. In gamma-actin gene-transfected substrains that have increased rates of gamma-actin synthesis, beta-actin synthesis is further reduced in a manner consistent with an autoregulatory mechanism, resulting in abnormal ratios of actin isoforms. Thus, both beta- and gamma-actin proteins can apparently regulate the synthesis of their coexpressed isoforms. In addition, decreased synthesis of normal beta-actin seems to correlate with a concomitant down-regulation of tropomyosin isoforms.

Actins↗
PubMed1987

Evolution of late H2A, H2B, and H4 histone genes of the sea urchin, Strongylocentrotus purpuratus.

Sea urchins possess several distinct sets of histone genes, including "early" genes, maximally active in cleavage and blastula stages, and "late" genes, active from the late blastula stage onwards. We determined the nucleotide sequences of six sea urchin (Strongylocentrotus purpuratus) late histone genes located on four genomic segments. Comparative analysis of these sequences identified several conserved elements in 5' flanking regions, including the sequences ATGPyATANTATA shared by all late genes and GGCGGGAAATTGAAAA shared by two late H4s. Comparisons of protein-coding sequences of late H4 and H2B genes with their early counterparts showed that silent sites have diverged to the theoretical maximum, indicating that early and late histone gene classes diverged at least 200 million years ago. Since extant echinoderms evolved from a common ancestor at about that time, it is likely that early and late histone gene sets are characteristic of all echinoderm groups. Amino acid sequences derived from nucleotide sequences of late H2A and H2B gistone genes differ substantially from amino acid sequences of their late counterparts. Most such differences are in highly mutable positions. A few, however, occur in positions that do not mutate frequently and thus may reflect functional differences between the early and late forms of the H2A and H2B proteins.

Amino Acid Sequence↗
PubMed1987

Alternative splicing generates variants in important functional domains of human slow skeletal troponin T.

We provide the first nucleotide sequence information for the slow isoform of troponin T (TnT). Sequence and hybridization analyses revealed that a single slow TnT gene present in the human genome gives rise to at least two different slow TnT variants by alternative splicing. The observed variations in slow TnT splicing generated major structural differences between the two corresponding slow TnT proteins in a domain that is likely to be involved in critical interactions with troponin C, troponin I, and tropomyosin in the thin filament. Corresponding variations have not been found for fast or for cardiac TnT. The comparison of splicing patterns for fast, cardiac, and slow TnT reveals that the splicing pattern for each isoform is unique. These features raise important questions of why and how all the individual members of the closely related TnT gene family developed such complex but different schemes of alternative splicing to create sets of variant proteins. This unusual familial trait is not known in any other muscle or nonmuscle multigene family.

Amino Acid Sequence↗
PubMed1987

Unusual structure, evolutionary conservation of non-coding sequences and numerous pseudogenes characterize the human H3.3 histone multigene family.

The genomic organization of the replication-independent, basally expressed, human H3.3 gene is atypical of traditional histone gene organization. The gene contains 3 introns totalling 7.8 kb and unusual direct repeats flank all three intron-exon splice junctions. The transcription initiation site was mapped by S1 nuclease protection analysis and confirms that cDNA clones previously reported were full length. Sequence similarities between regions at the 5' and 3' termini of this human gene and a chicken H3.3 gene lead us to propose that either the previous assignments of termini of the chicken gene are in error, or there are alternative transcription start and polyadenylation sites. The 85% base matching of human and chicken H3.3 3'UTR sequences for 520 bases is unprecedented among homolog 3'UTR segments, especially considering that these species are separated by over 250 Myr of evolution. We also present the sequence of three related processed human H3.3 pseudogenes and provide evidence demonstrating that most of the 20 to 30 copies of the H3.3 gene within the human genome are in fact processed pseudogenes.

Amino Acid Sequence↗
PubMed1987

A human beta-actin expression vector system directs high-level accumulation of antisense transcripts.

We have constructed a mammalian expression vector consisting of 3 kilobases of the human beta-actin gene 5' flanking sequence plus 5' untranslated region and intervening sequence 1 linked at the 3' splice site to a short DNA polylinker sequence containing unique Sal I, HindIII, and BamHI restriction endonuclease sites followed by a simian virus 40 (SV40) polyadenylylation signal. Two derivatives, containing the selection markers obtained from pSV2gpt or pSV2neo, were also generated. We find that the promoter activity of this vector is a great or greater than that of the SV40 early promoter in a variety of human and rodent cells. The vector was used to generate gamma-actin and beta-tubulin antisense transcripts in human fibroblast cell lines. The antisense transcripts accumulate to levels comparable with that of the highly abundant gamma-actin and beta-tubulin mRNAs.

Actins↗