Rat metallothionein-1 structural gene and three pseudogenes, one of which contains 5'-regulatory sequences

1986 ◽  
Vol 6 (1) ◽  
pp. 302-314
Author(s):  
R D Andersen ◽  
B W Birren ◽  
S J Taplitz ◽  
H R Herschman
Keyword(s):  
Rna Polymerase Ii ◽  
Structural Gene ◽  
Transcription Initiation ◽  
Metal Ion ◽  
Consensus Sequence ◽  
Initiation Site ◽  
Transcription Initiation Site ◽  
Regulatory Sequences ◽  
Base Pairs ◽  
Nuclease Mapping

As shown by Southern blot analysis, the metallothionein-1 (MT-1) genes in rats comprise a multigene family. We present the sequence of the MT-1 structural gene and compare its features with other metallothionein genes. Three MT-1 pseudogenes which we sequenced apparently arose by reverse transcription of processed mRNA transcripts. Two of these, MT-1 psi a and MT-1 psi c, are retrogenes which derive from the MT-1 mRNA, having diverged from the MT-1 gene 6.9 and 2.6 million years ago, respectively. The third, MT-1 psi b, differs from the MT-1 cDNA by only three nucleotide alterations. Surprisingly, MT-1 psi b also preserves sequence homology for 142 base pairs 5' to the transcription initiation site of the parent gene; it contains a promoter sequence sufficient for specifying metal ion induction. We identified, by S1 nuclease mapping, an RNA polymerase II initiation site 432 base pairs 5' of the MT-1 transcription initiation site of the MT-1 structural gene which could explain the formation of the mRNA precursor to this pseudogene. We were unable to detect MT-1 psi b transcripts, either in liver tissue or after transfection. We conclude that the absence of detectable transcripts from this pseudogene is due to either a reduced level of transcription or the formation of unstable transcripts as a consequence of the lack of a consensus sequence normally found 3' of transcription termination in the MT-1 structural gene.

scholarly journals Rat metallothionein-1 structural gene and three pseudogenes, one of which contains 5'-regulatory sequences.

1986 ◽  
Vol 6 (1) ◽  
pp. 302-314 ◽  
Author(s):  
R D Andersen ◽  
B W Birren ◽  
S J Taplitz ◽  
H R Herschman
Keyword(s):  
Rna Polymerase Ii ◽  
Structural Gene ◽  
Transcription Initiation ◽  
Metal Ion ◽  
Consensus Sequence ◽  
Initiation Site ◽  
Transcription Initiation Site ◽  
Regulatory Sequences ◽  
Base Pairs ◽  
Nuclease Mapping

As shown by Southern blot analysis, the metallothionein-1 (MT-1) genes in rats comprise a multigene family. We present the sequence of the MT-1 structural gene and compare its features with other metallothionein genes. Three MT-1 pseudogenes which we sequenced apparently arose by reverse transcription of processed mRNA transcripts. Two of these, MT-1 psi a and MT-1 psi c, are retrogenes which derive from the MT-1 mRNA, having diverged from the MT-1 gene 6.9 and 2.6 million years ago, respectively. The third, MT-1 psi b, differs from the MT-1 cDNA by only three nucleotide alterations. Surprisingly, MT-1 psi b also preserves sequence homology for 142 base pairs 5' to the transcription initiation site of the parent gene; it contains a promoter sequence sufficient for specifying metal ion induction. We identified, by S1 nuclease mapping, an RNA polymerase II initiation site 432 base pairs 5' of the MT-1 transcription initiation site of the MT-1 structural gene which could explain the formation of the mRNA precursor to this pseudogene. We were unable to detect MT-1 psi b transcripts, either in liver tissue or after transfection. We conclude that the absence of detectable transcripts from this pseudogene is due to either a reduced level of transcription or the formation of unstable transcripts as a consequence of the lack of a consensus sequence normally found 3' of transcription termination in the MT-1 structural gene.

scholarly journals Expression of the murine alpha B-crystallin gene is not restricted to the lens.

1989 ◽  
Vol 9 (3) ◽  
pp. 1083-1091 ◽  
Author(s):  
R A Dubin ◽  
E F Wawrousek ◽  
J Piatigorsky
Keyword(s):  
Skeletal Muscle ◽  
Transcription Initiation ◽  
Germ Line ◽  
Initiation Site ◽  
Regulatory Sequences ◽  
Base Pairs ◽  
In Vitro System ◽  
Endogenous Gene ◽  
Efficient Expression

The murine alpha B-crystallin gene was cloned and its expression was examined. In the mouse, significant levels of alpha B-crystallin RNA were detected not only in lens but also in heart, skeletal muscle, kidney, and lung; low and trace levels were detected in brain and spleen, respectively. The RNA species in lung, brain, and spleen was 400 to 500 bases larger than that in the other tissues. Transcription in lens, heart, skeletal muscle, kidney, and brain initiated at the same position. A mouse alpha B-crystallin mini-gene was constructed and was introduced into the germ line of mice, and its expression was demonstrated to parallel that of the endogenous gene. Transgene RNA was always detected in lens, heart, and skeletal muscle, while expression in kidney and lung was variable; it remains uncertain whether there is transgene expression in brain and spleen. These results demonstrate that regulatory sequences controlling expression of the alpha B-crystallin gene lie between sequences 666 base pairs upstream of the transcription initiation site and 2.4 kilobase pairs downstream of the poly(A) addition site and are not located within the introns. Transfection studies with a series of alpha B-crystallin mini-gene deletion mutants revealed that sequences between positions -222 and -167 were required for efficient expression in primary embryonic chick lens cells; sequences downstream of the poly(A) addition signal were dispensable for expression in this in vitro system.

scholarly journals c-mos expression in mouse oocytes is controlled by initiator-related sequences immediately downstream of the transcription initiation site

1991 ◽  
Vol 11 (10) ◽  
pp. 5190-5196
Author(s):  
S K Pal ◽  
S S Zinkel ◽  
A A Kiessling ◽  
G M Cooper
Keyword(s):  
Transcription Start Site ◽  
Transcription Initiation ◽  
Promoter Activity ◽  
Consensus Sequence ◽  
Initiation Site ◽  
Transcription Initiation Site ◽  
Transcriptional Level ◽  
Start Site ◽  
Transcription Start ◽  
Mouse Oocytes

We have employed transient expression assays to analyze the sequences that direct c-mos transcription in mouse oocytes. Plasmids containing the chloramphenicol acetyltransferase (CAT) gene fused to either a 2.4-kb or a 731-bp fragment from the 5'-flanking region of c-mos produced similar levels of CAT activity when injected into nuclei of growing oocytes. BAL 31 deletions revealed that sequences up to 20 bp upstream of the major transcription start site could be removed without any significant loss of CAT activity. Promoter activity only decreased when these deletions closely approached the transcription start site, which was mapped at 53 nucleotides upstream of the first ATG in the c-mos open reading frame. On the other hand, deletion of sequences within 20 nucleotides downstream of the transcription initiation site resulted in a 10-fold reduction in CAT expression. A similar decrease in promoter activity was observed as a result of point mutations in these 5' untranslated sequences. Thus, sequences immediately downstream of the transcription start site, including a consensus sequence (PyPyCAPyPyPyPyPy) present in the initiator elements of several genes, appear to regulate c-mos expression in mouse oocytes. Reverse transcription-polymerase chain reaction analysis of RNA from injected oocytes showed that this regulation is manifest at the transcriptional level. Expression of c-mos in mouse oocytes thus appears to be directed by a simple promoter consisting only of sequences immediately surrounding the transcription start site, including an initiator element in the untranslated leader.

scholarly journals Structure and transcriptional regulation of the Nat2 gene encoding for the drug-metabolizing enzyme arylamine N-acetyltransferase type 2 in mice

2003 ◽  
Vol 375 (3) ◽  
pp. 593-602 ◽  
Author(s):  
Sotiria BOUKOUVALA ◽  
Naomi PRICE ◽  
Kathryn E. PLANT ◽  
Edith SIM
Keyword(s):  
Transcriptional Regulation ◽  
Transcription Initiation ◽  
Consensus Sequence ◽  
Simian Virus 40 ◽  
Initiation Site ◽  
Cellular Protein ◽  
Transcription Initiation Site ◽  
Gene Promoters ◽  
Coding Region ◽  
Nat2 Gene

Arylamine N-acetyltransferases (NATs) are polymorphic enzymes, well-known for their role in the metabolism of drugs and carcinogens. Mice have three NAT isoenzymes, of which NAT2 is postulated to be involved in endogenous, as well as xenobiotic, metabolism. To understand expression of the murine Nat2 gene, we have analysed its structure and transcriptional regulation. We have accurately mapped the transcription initiation site 6.5 kb upstream of the coding region of the gene, adjacent to a recently described non-coding exon. Transcription was demonstrated to start from this region in embryonic and adult liver, spleen, submaxillary gland, kidney, brain, thymus, lung and placenta, but not in the heart. Database searches and analyses of cDNA by PCR suggested alternative splicing of the single 6.2 kb intron of Nat2, and determined the position of the polyadenylation signal at 0.44 kb downstream of the coding region of the gene. Examination of the 13 kb sequence flanking the coding and non-coding exons of Nat2 revealed a single promoter, located close to the transcription-initiation site, and indicated regions likely to harbour control elements. The Nat2 promoter consists of an atypical TATA box and a Sp1 [SV40 (simian virus 40) protein 1] box identical with that found in many housekeeping gene promoters. Activity of the Nat2 promoter was severely reduced by deletion or mutation of either of these two elements, whereas the region of the Sp1 box bound cellular protein and resisted DNase I digestion. Finally, the ability of the promoter region to bind cellular protein was reduced by competition with oligonucleotides bearing the Sp1 consensus sequence.

scholarly journals Isolation and characterization of the human diacylglycerol kinase gene

1993 ◽  
Vol 294 (2) ◽  
pp. 443-449 ◽  
Author(s):  
K Fujikawa ◽  
S Imai ◽  
F Sakane ◽  
H Kanoh
Keyword(s):  
Transcription Initiation ◽  
Diacylglycerol Kinase ◽  
Initiation Site ◽  
Transcription Initiation Site ◽  
Jurkat Cells ◽  
Upstream Region ◽  
Negative Element ◽  
Isolation And Characterization ◽  
Flanking Region ◽  
Nuclease Mapping

The 80 kDa diacylglycerol kinase (DGK) is abundantly expressed in oligodendrocytes and lymphocytes but not to a detectable extent in other cells such as neurons and hepatocytes. As an initial attempt to delineate the mechanism of the transcriptional control of the DGK gene, we have cloned from a human genomic library a 22 kb genomic fragment. The genomic clone consists of the 5′-flanking region and 17 exons coding for approx. 53% of the total exons of human DGK, including those encoding EF-hand and zinc-finger regions. The translation initiation site is located in the second exon. S1 nuclease mapping and primer extension analysis of the human DGK mRNA identified a major transcription initiation site (position +1) at 264 bp upstream from the initiator ATG. In the 5′-flanking sequence we detected a single GC box at -35 but no canonical TATA and CAAT sequences. However, the sequence starting from the cap site (AGTTCCTGCCA) is very similar to the initiator element that specifies the transcription initiation site of some housekeeping genes. In addition, the 5′-upstream region contains several putative cis-elements. Jurkat and HepG2 cells were transfected with various 5′-deletion mutants of the upstream region fused to the structural gene of chloramphenicol acetyltransferase (CAT). The CAT assay revealed that among constructs containing up to 3.4 kb of the 5′-flanking region, a fragment of 263 bp from the transcription initiation site contains a basic promoter that is active in both types of cells. Moreover, the region between -263 and -850 contains a negative element that is active in HepG2 but not in Jurkat cells. This negative element may, at least in part, be responsible for the cell type-specific expression of the DGK gene.

Expression of the murine alpha B-crystallin gene is not restricted to the lens

1989 ◽  
Vol 9 (3) ◽  
pp. 1083-1091
Author(s):  
R A Dubin ◽  
E F Wawrousek ◽  
J Piatigorsky
Keyword(s):  
Skeletal Muscle ◽  
Transcription Initiation ◽  
Germ Line ◽  
Initiation Site ◽  
Regulatory Sequences ◽  
Base Pairs ◽  
In Vitro System ◽  
Endogenous Gene ◽  
Efficient Expression

The murine alpha B-crystallin gene was cloned and its expression was examined. In the mouse, significant levels of alpha B-crystallin RNA were detected not only in lens but also in heart, skeletal muscle, kidney, and lung; low and trace levels were detected in brain and spleen, respectively. The RNA species in lung, brain, and spleen was 400 to 500 bases larger than that in the other tissues. Transcription in lens, heart, skeletal muscle, kidney, and brain initiated at the same position. A mouse alpha B-crystallin mini-gene was constructed and was introduced into the germ line of mice, and its expression was demonstrated to parallel that of the endogenous gene. Transgene RNA was always detected in lens, heart, and skeletal muscle, while expression in kidney and lung was variable; it remains uncertain whether there is transgene expression in brain and spleen. These results demonstrate that regulatory sequences controlling expression of the alpha B-crystallin gene lie between sequences 666 base pairs upstream of the transcription initiation site and 2.4 kilobase pairs downstream of the poly(A) addition site and are not located within the introns. Transfection studies with a series of alpha B-crystallin mini-gene deletion mutants revealed that sequences between positions -222 and -167 were required for efficient expression in primary embryonic chick lens cells; sequences downstream of the poly(A) addition signal were dispensable for expression in this in vitro system.

scholarly journals c-mos expression in mouse oocytes is controlled by initiator-related sequences immediately downstream of the transcription initiation site.

1991 ◽  
Vol 11 (10) ◽  
pp. 5190-5196 ◽  
Author(s):  
S K Pal ◽  
S S Zinkel ◽  
A A Kiessling ◽  
G M Cooper
Keyword(s):  
Transcription Start Site ◽  
Transcription Initiation ◽  
Promoter Activity ◽  
Consensus Sequence ◽  
Initiation Site ◽  
Transcription Initiation Site ◽  
Transcriptional Level ◽  
Start Site ◽  
Transcription Start ◽  
Mouse Oocytes

We have employed transient expression assays to analyze the sequences that direct c-mos transcription in mouse oocytes. Plasmids containing the chloramphenicol acetyltransferase (CAT) gene fused to either a 2.4-kb or a 731-bp fragment from the 5'-flanking region of c-mos produced similar levels of CAT activity when injected into nuclei of growing oocytes. BAL 31 deletions revealed that sequences up to 20 bp upstream of the major transcription start site could be removed without any significant loss of CAT activity. Promoter activity only decreased when these deletions closely approached the transcription start site, which was mapped at 53 nucleotides upstream of the first ATG in the c-mos open reading frame. On the other hand, deletion of sequences within 20 nucleotides downstream of the transcription initiation site resulted in a 10-fold reduction in CAT expression. A similar decrease in promoter activity was observed as a result of point mutations in these 5' untranslated sequences. Thus, sequences immediately downstream of the transcription start site, including a consensus sequence (PyPyCAPyPyPyPyPy) present in the initiator elements of several genes, appear to regulate c-mos expression in mouse oocytes. Reverse transcription-polymerase chain reaction analysis of RNA from injected oocytes showed that this regulation is manifest at the transcriptional level. Expression of c-mos in mouse oocytes thus appears to be directed by a simple promoter consisting only of sequences immediately surrounding the transcription start site, including an initiator element in the untranslated leader.

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