scholarly journals Structure and tissue-specific expression of the human metallothionein IB gene.

1986 ◽  
Vol 6 (6) ◽  
pp. 2149-2157 ◽  
Author(s):  
A Heguy ◽  
A West ◽  
R I Richards ◽  
M Karin
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Transcription Initiation ◽  
Cell Types ◽  
Initiation Site ◽  
Predicted Amino Acid Sequence ◽  
Functional Protein ◽  
Specific Expression ◽  
Cis Acting ◽  
High Level

The human metallothionein (MT) IB gene (hMT-IB) is located in a region of human DNA containing at least four tandemly arranged MT genes. As deduced from its sequence, hMT-IB is likely to encode a functional protein. However, the predicted amino acid sequence differed from the hMT-I amino acid sequence in four positions. Most remarkable was the presence of an additional cysteine. Like other MT genes, hMT-IB has at least two copies of the metal-responsive element upstream from the transcription initiation site. These elements probably are responsible for the metal responsiveness of the hMT-IB promoter, leading to inducible expression of fused heterologous genes. Unlike the hMT-IIA and hMT-IA genes described previously, which are expressed in many different cell types, a high level of expression of the endogenous hMT-IB gene could be detected only in human hepatoma and renal carcinoma cell lines. Therefore, this is the first MT gene described which exhibits tissue specificity of expression. This specificity is controlled by a cis-acting mechanism involving methylation, since incubation of nonexpressing cells with an inhibitor of DNA methylation led to activation of the hMT-IB gene. In support of this notion, we found that the 5' flanking region of the hMT-IB gene was highly methylated in HeLa cells, a nonexpressing cell type, but it was not methylated in a hepatoma (expressing) cell line.

Structure and tissue-specific expression of the human metallothionein IB gene

1986 ◽  
Vol 6 (6) ◽  
pp. 2149-2157
Author(s):  
A Heguy ◽  
A West ◽  
R I Richards ◽  
M Karin
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Transcription Initiation ◽  
Cell Types ◽  
Initiation Site ◽  
Predicted Amino Acid Sequence ◽  
Functional Protein ◽  
Specific Expression ◽  
Cis Acting ◽  
High Level

The human metallothionein (MT) IB gene (hMT-IB) is located in a region of human DNA containing at least four tandemly arranged MT genes. As deduced from its sequence, hMT-IB is likely to encode a functional protein. However, the predicted amino acid sequence differed from the hMT-I amino acid sequence in four positions. Most remarkable was the presence of an additional cysteine. Like other MT genes, hMT-IB has at least two copies of the metal-responsive element upstream from the transcription initiation site. These elements probably are responsible for the metal responsiveness of the hMT-IB promoter, leading to inducible expression of fused heterologous genes. Unlike the hMT-IIA and hMT-IA genes described previously, which are expressed in many different cell types, a high level of expression of the endogenous hMT-IB gene could be detected only in human hepatoma and renal carcinoma cell lines. Therefore, this is the first MT gene described which exhibits tissue specificity of expression. This specificity is controlled by a cis-acting mechanism involving methylation, since incubation of nonexpressing cells with an inhibitor of DNA methylation led to activation of the hMT-IB gene. In support of this notion, we found that the 5' flanking region of the hMT-IB gene was highly methylated in HeLa cells, a nonexpressing cell type, but it was not methylated in a hepatoma (expressing) cell line.

scholarly journals Structure and tissue-specific expression of the Drosophila melanogaster organellar-type Ca2+-ATPase gene

1995 ◽  
Vol 310 (3) ◽  
pp. 757-763 ◽  
Author(s):  
A Magyar ◽  
E Bakos ◽  
A Váradi
Keyword(s):  
Drosophila Melanogaster ◽  
Transcription Initiation ◽  
Initiation Site ◽  
Drosophila Genome ◽  
Coding Region ◽  
Specific Expression ◽  
S1 Nuclease ◽  
Protein Coding ◽  
Atpase Gene ◽  
High Level

A 14 kb genomic clone covering the organellar-type Ca(2+)-ATPase gene of Drosophila melanogaster has been isolated and characterized. The sequence of a 7132 bp region extending from 1.1 kb 5′ upstream of the initiation ATG codon over the polyadenylation signal at the 3′ end has been determined. The gene consists of nine exons including one with an exceptional size of 2172 bp representing 72% of the protein coding region. Introns are relatively small (< 100 bp) except for the 3′ intron which has a size of 2239 bp, an exceptionally large size among Drosophila introns. Five of the introns are in the same positions in Drosophila, Artemia and rabbit SERCA1 Ca(2+)-ATPase genes. There is only one organellar-type Ca(2+)-ATPase gene in the Drosophila genome, as was shown by Southern-blot analysis [Váradi, Gilmore-Hebert and Benz (1989) FEBS Lett. 258, 203-207] and by chromosomal localization [Magyar and Váradi (1990) Biochem. Biophys. Res. Commun. 173, 872-877]. Primer extension and S1-nuclease assays revealed a potential transcription initiation site 876 bp upstream of the translation initiation ATG with a TATA-box 23 bp upstream of this site. Analysis of the 5′ region of the Drosophila organellar-type Ca(2+)-ATPase gene suggests the presence of potential recognition sequences of various muscle-specific transcription factors and shows a region with remarkable similarity to that in the rabbit SERCA2 gene. The tissue distribution of expression of the organellar-type Ca(2+)-ATPase gene has been studied by in situ RNA-RNA hybridization on microscopic sections. A low mRNA abundance can be detected in each tissue of adult flies, suggesting a housekeeping function for the gene. On the other hand a pronounced tissue specificity of expression has also been found as the organellar-type Ca(2+)-ATPase is expressed at a very high level in cell bodies of the central nervous system and in various muscles.

scholarly journals Serine and threonine catabolism in Saccharomyces cerevisiae: the CHA1 polypeptide is homologous with other serine and threonine dehydratases.

Genetics ◽  
1992 ◽  
Vol 131 (3) ◽  
pp. 531-539 ◽  
Author(s):  
C Bornaes ◽  
J G Petersen ◽  
S Holmberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Transcription Initiation ◽  
Open Reading Frame ◽  
Predicted Amino Acid Sequence ◽  
S1 Nuclease ◽  
Reading Frame ◽  
Threonine Dehydratase ◽  
Nuclease Mapping

Abstract The catabolic L-serine (L-threonine) dehydratase of Saccharomyces cerevisiae allows the yeast to grow on media with L-serine or L-threonine as sole nitrogen source. Previously we have cloned the CHA1 gene by complementation of a mutant, cha1, lacking the dehydratase activity. Here we present the DNA sequence of a 1,766-bp fragment of the CHA1 region encompassing an open reading frame of 1080 bp. Comparison of the predicted amino acid sequence of the CHA1 polypeptide with that of other serine/threonine dehydratases revealed several blocks of sequence homology. Thus, the amino acid sequence of rat liver serine dehydratase (SDH2) and the CHA1 polypeptide are 44% homologous allowing for conservative substitutions, while 36% similarity is found between the catabolic threonine dehydratase (tdcB) of Escherichia coli and the CHA1 protein. This strongly suggests that CHA1 is the structural gene for the yeast catabolic serine (threonine) dehydratase. S1-nuclease mapping of the CHA1 mRNA ends showed a major transcription initiation site corresponding to an untranslated leader of about 19 nucleotides, while a major polyadenylation site was located about 86 nucleotides downstream from the open reading frame. Furthermore, we have mapped the chromosomal position of the CHA1 gene to less than 0.5 kb centromere proximal to HML on the left arm of chromosome III.

scholarly journals The transcriptional tissue specificity of the human proα1(I) collagen gene is determined by a negative cis-regulatory element in the promoter

1992 ◽  
Vol 286 (1) ◽  
pp. 179-185 ◽  
Author(s):  
C P Simkevich ◽  
J P Thompson ◽  
H Poppleton ◽  
R Raghow
Keyword(s):  
Tissue Specificity ◽  
Regulatory Element ◽  
Mesenchymal Cell ◽  
Cell Types ◽  
Regulatory Elements ◽  
Negative Influence ◽  
Collagen Gene ◽  
Specific Expression ◽  
Cis Acting ◽  
First Intron

The transcriptional activity of plasmid pCOL-KT, in which human pro alpha 1 (I) collagen gene upstream sequences up to -804 and most of the first intron (+474 to +1440) drive expression of the chloramphenicol acetyltransferase (CAT) gene [Thompson, Simkevich, Holness, Kang & Raghow (1991) J. Biol. Chem. 266, 2549-2556], was tested in a number of mesenchymal and non-mesenchymal cells. We observed that pCOL-KT was readily expressed in fibroblasts of human (IMR-90 and HFL-1), murine (NIH 3T3) and avian (SL-29) origin and in a human rhabdomyosarcoma cell line (A204), but failed to be expressed in human erythroleukaemia (K562) and rat pheochromocytoma (PC12) cells, indicating that the regulatory elements required for appropriate tissue-specific expression of the human pro alpha 1 (I) collagen gene were present in pCOL-KT. To delineate the nature of cis-acting sequences which determine the tissue specificity of pro alpha 1 (I) collagen gene expression, functional consequences of deletions in the promoter and first intron of pCOL-KT were tested in various cell types by transient expression assays. Cis elements in the promoter-proximal and intronic sequences displayed either a positive or a negative influence depending on the cell type. Thus deletion of fragments using EcoRV (nt -625 to -442 deleted), XbaI (-804 to -331) or SstII (+670 to +1440) resulted in 2-10-fold decreased expression in A204 and HFL-1 cells. The negative influences of deletions in the promoter-proximal sequences was apparently considerably relieved by deleting sequences in the first intron, and the constructs containing the EcoRV/SstII or XbaI/SstII double deletions were expressed to a much greater extent than either of the single deletion constructs. In contrast, the XbaI* deletion (nt -804 to -609), either alone or in combination with the intronic deletion, resulted in very high expression in all cells regardless of their collagen phenotype; the XbaI*/(-SstII) construct, which contained the intronic SstII fragment (+670 to +1440) in the reverse orientation, was not expressed in either mesenchymal or nonmesenchymal cells. Based on these results, we conclude that orientation-dependent interactions between negatively acting 5′-upstream sequences and the first intron determine the mesenchymal cell specificity of human pro alpha 1 (I) collagen gene transcription.

scholarly journals Characterization of Gmhsp26-A, a stress gene encoding a divergent heat shock protein of soybean: heavy-metal-induced inhibition of intron processing.

1988 ◽  
Vol 8 (3) ◽  
pp. 1113-1122 ◽  
Author(s):  
E Czarnecka ◽  
R T Nagao ◽  
J L Key ◽  
W B Gurley
Keyword(s):  
Amino Acid ◽  
Heat Shock ◽  
Amino Acid Sequence ◽  
Genomic Sequence ◽  
Stress Protein ◽  
Initiation Site ◽  
Gene Encoding ◽  
S1 Nuclease ◽  
Soybean Seedlings ◽  
Nuclease Mapping

We determined the DNA sequence and mapped the corresponding transcripts of a genomic clone containing the Gmhsp26-A gene of soybean. This gene is homologous to the previously characterized cDNA clone pCE54 (E. Czarnecka, L. Edelman, F. Schöffl, and J. L. Key, Plant Mol. Biol. 3:45-58, 1984) and is expressed in response to a wide variety of physiological stresses including heat shock (HS). S1 nuclease mapping of transcripts and a comparison of the cDNA sequence with the genomic sequence indicated the presence of a soybean seedlings with either CdCl2 or CuSO4. Analysis of the 5' termini of transcripts indicated the presence of one major and at least two minor start sites. In each case, initiation occurred 27 to 30 base pairs downstream from a TATA-like motif, and thus each initiation site appears to be promoted by the activity of a separate subpromoter. The three subpromoters are all associated with sequences showing low homology to the HS consensus element of Drosophila melanogaster HS genes and are differentially induced in response to various stresses. Within the carboxyl-terminal half of the protein, hydropathy analysis of the deduced amino acid sequence indicated a high degree of relatedness to the small HS proteins. A comparison of the primary amino acid sequence of hsp26-A with sequences of the small HS proteins suggested that this stress protein is highly diverged and may therefore be specialized for stress adaptation in soybean.

Identification of upstream and intragenic regulatory elements that confer cell-type-restricted and differentiation-specific expression on the muscle creatine kinase gene

1988 ◽  
Vol 8 (7) ◽  
pp. 2896-2909 ◽  
Author(s):  
E A Sternberg ◽  
G Spizz ◽  
W M Perry ◽  
D Vizard ◽  
T Weil ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Regulatory Element ◽  
Simian Virus 40 ◽  
Muscle Cells ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Simian Virus ◽  
Cell Type ◽  
Specific Expression ◽  
Developing Muscle

Terminal differentiation of skeletal myoblasts is accompanied by induction of a series of tissue-specific gene products, which includes the muscle isoenzyme of creatine kinase (MCK). To begin to define the sequences and signals involved in MCK regulation in developing muscle cells, the mouse MCK gene has been isolated. Sequence analysis of 4,147 bases of DNA surrounding the transcription initiation site revealed several interesting structural features, some of which are common to other muscle-specific genes and to cellular and viral enhancers. To test for sequences required for regulated expression, a region upstream of the MCK gene from -4800 to +1 base pairs, relative to the transcription initiation site, was linked to the coding sequences of the bacterial chloramphenicol acetyltransferase (CAT) gene. Introduction of this MCK-CAT fusion gene into C2 muscle cells resulted in high-level expression of CAT activity in differentiated myotubes and no detectable expression in proliferating undifferentiated myoblasts or in nonmyogenic cell lines. Deletion mutagenesis of sequences between -4800 and the transcription start site showed that the region between -1351 and -1050 was sufficient to confer cell type-specific and developmentally regulated expression on the MCK promoter. This upstream regulatory element functioned independently of position, orientation, or distance from the promoter and therefore exhibited the properties of a classical enhancer. This upstream enhancer also was able to confer muscle-specific regulation on the simian virus 40 promoter, although it exhibited a 3- to 5-fold preference for its own promoter. In contrast to the cell type- and differentiation-specific expression of the upstream enhancer, the MCK promoter was able to function in myoblasts and myotubes and in nonmyogenic cell lines when combined with the simian virus 40 enhancer. An additional positive regulatory element was identified within the first intron of the MCK gene. Like the upstream enhancer, this intragenic element functioned independently of position, orientation, and distance with respect to the MCK promoter and was active in differentiated myotubes but not in myoblasts. These results demonstrate that expression of the MCK gene in developing muscle cells is controlled by complex interactions among multiple upstream and intragenic regulatory elements that are functional only in the appropriate cellular context.

Conserved DNA binding and self-association domains of the Drosophila zeste protein

1992 ◽  
Vol 12 (2) ◽  
pp. 598-608
Author(s):  
J D Chen ◽  
C S Chan ◽  
V Pirrotta
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Amino Acid Sequence ◽  
Coiled Coil ◽  
Helical Structure ◽  
Binding Activity ◽  
Binding Domain ◽  
Drosophila Virilis ◽  
Predicted Amino Acid Sequence ◽  
Dna Binding Domain

The zeste gene product is involved in two types of genetic effects dependent on chromosome pairing: transvection and the zeste-white interaction. Comparison of the predicted amino acid sequence with that of the Drosophila virilis gene shows that several blocks of amino acid sequence have been very highly conserved. One of these regions corresponds to the DNA binding domain. Site-directed mutations in this region indicate that a sequence resembling that of the homeodomain DNA recognition helix is essential for DNA binding activity. The integrity of an amphipathic helical region is also essential for binding activity and is likely to be responsible for dimerization of the DNA binding domain. Another very strongly conserved domain of zeste is the C-terminal region, predicted to form a long helical structure with two sets of heptad repeats that constitute two long hydrophobic ridges at opposite ends and on opposite faces of the helix. We show that this domain is responsible for the extensive aggregation properties of zeste that are required for its role in transvection phenomena. A model is proposed according to which the hydrophobic ridges induce the formation of open-ended coiled-coil structures holding together many hundreds of zeste molecules and possibly anchoring these complexes to other nuclear structures.

scholarly journals Characterization of a Novel Plasmid-Mediated Cephalosporinase (CMY-9) and Its Genetic Environment in an Escherichia coli Clinical Isolate

2002 ◽  
Vol 46 (8) ◽  
pp. 2427-2434 ◽  
Author(s):  
Yohei Doi ◽  
Naohiro Shibata ◽  
Keigo Shibayama ◽  
Kazunari Kamachi ◽  
Hiroshi Kurokawa ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Gene Cassette ◽  
Single Amino Acid ◽  
Common Region ◽  
Class 1 Integron ◽  
Class 1 ◽  
The Common ◽  
High Level

ABSTRACT An Escherichia coli strain, HKYM68, which showed resistance to broad-spectrum cephalosporins was isolated from a sputum specimen in Japan. The high-level resistance of the strain to ceftazidime, cefpirome, and moxalactam was carried by a self-transferable plasmid. The β-lactamase gene responsible for the resistance was cloned and sequenced. The deduced amino acid sequence of this gene product, CMY-9, had a single amino acid substitution (E85D), the residue reported to be part of the recognition site for the R1 side chain of β-lactams, compared with the amino acid sequence of CMY-8 and also had 78% identity with the amino acid sequence of CepH, a chromosomal cephalosporinase of Aeromonas hydrophila. A sul1-type class 1 integron containing an aacA1-orfG gene cassette was identified upstream of bla CMY-9 and ended with a truncated 3′ conserved segment. The following 2.1 kb was almost identical to the common region of integrons In6 and In7 and the integron of pSAL-1, except that orf513 encoding a putative transposase was identified instead of orf341 due to addition of a single nucleotide. bla CMY-9 was closely located downstream of the end of the common region. These observations are indicative of the exogenous derivation of bla CMY-9 from some environmental microorganisms such as aeromonads.

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