Sequence requirements for transcriptional arrest in exon 1 of the murine adenosine deaminase gene

1990 ◽  
Vol 10 (4) ◽  
pp. 1484-1491
Author(s):  
V Ramamurthy ◽  
M C Maa ◽  
M L Harless ◽  
D A Wright ◽  
R E Kellems
Keyword(s):  
Adenosine Deaminase ◽  
Transcription Initiation ◽  
Substantial Reduction ◽  
Initiation Site ◽  
Xenopus Laevis Oocytes ◽  
Functional Assay ◽  
Base Pairs ◽  
Base Pair Fragment ◽  
Exon 1 ◽  
Translation Initiation Codon

We have previously shown that a transcription arrest site near the 5' end of the murine adenosine deaminase (ADA) gene is significantly involved in the regulation of ADA gene expression. To facilitate the analysis of this transcription arrest site, we have analyzed the transcription products from cloned ADA gene fragments injected into Xenopus laevis oocytes. When genomic fragments spanning the 5' end of the ADA gene were injected into oocytes, a 96-nucleotide (nt) ADA RNA was the major transcription product. The 5' end of this RNA mapped to the transcription initiation site for the ADA gene, and its 3' terminus mapped 7 nt downstream of the translation initiation codon within exon 1. A 300-base-pair fragment of genomic DNA spanning the 5' end of the ADA gene was sufficient to generate the 96-nt transcript which accounted for approximately one-half of the transcription products from injected templates. Deletion of a segment of approximately 65 base pairs, located immediately downstream of the 3' terminus of the 96-nt transcript, resulted in a substantial reduction in the synthesis of the 96-nt transcript and a corresponding increase in the production of larger transcripts. These studies show that the transcriptional apparatus of X. laevis oocytes responds to the transcription arrest site associated with exon 1 of the murine ADA gene and that oocyte injections provide a convenient functional assay for additional mechanistic studies.

scholarly journals Sequence requirements for transcriptional arrest in exon 1 of the murine adenosine deaminase gene.

1990 ◽  
Vol 10 (4) ◽  
pp. 1484-1491 ◽  
Author(s):  
V Ramamurthy ◽  
M C Maa ◽  
M L Harless ◽  
D A Wright ◽  
R E Kellems
Keyword(s):  
Adenosine Deaminase ◽  
Transcription Initiation ◽  
Substantial Reduction ◽  
Initiation Site ◽  
Xenopus Laevis Oocytes ◽  
Functional Assay ◽  
Base Pairs ◽  
Base Pair Fragment ◽  
Exon 1 ◽  
Translation Initiation Codon

We have previously shown that a transcription arrest site near the 5' end of the murine adenosine deaminase (ADA) gene is significantly involved in the regulation of ADA gene expression. To facilitate the analysis of this transcription arrest site, we have analyzed the transcription products from cloned ADA gene fragments injected into Xenopus laevis oocytes. When genomic fragments spanning the 5' end of the ADA gene were injected into oocytes, a 96-nucleotide (nt) ADA RNA was the major transcription product. The 5' end of this RNA mapped to the transcription initiation site for the ADA gene, and its 3' terminus mapped 7 nt downstream of the translation initiation codon within exon 1. A 300-base-pair fragment of genomic DNA spanning the 5' end of the ADA gene was sufficient to generate the 96-nt transcript which accounted for approximately one-half of the transcription products from injected templates. Deletion of a segment of approximately 65 base pairs, located immediately downstream of the 3' terminus of the 96-nt transcript, resulted in a substantial reduction in the synthesis of the 96-nt transcript and a corresponding increase in the production of larger transcripts. These studies show that the transcriptional apparatus of X. laevis oocytes responds to the transcription arrest site associated with exon 1 of the murine ADA gene and that oocyte injections provide a convenient functional assay for additional mechanistic studies.

CHARACTERIZATION OF THE AUXIN SYNTHESIS GENES OF ERWINIA HERBICOLA PV. GYPSOPHILAE

1997 ◽  
Vol 45 (4) ◽  
pp. 279-284 ◽  
Author(s):  
Yedidya Gafni ◽  
Shulamit Manulis ◽  
Talya Kunik ◽  
Amnon Lichter ◽  
Isaac Barash ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Initiation Site ◽  
Transcription Unit ◽  
Open Reading Frames ◽  
Virulence Plasmid ◽  
Base Pairs ◽  
Erwinia Herbicola ◽  
E Coli ◽  
Auxin Synthesis ◽  
Translation Initiation Codon

We present the DNA sequence of some 3590 base pairs from the virulence plasmid of the crown-gall-forming bacteriumErwinia herbicolapv.gypsophilaestrain PD713. This region includes the entire transcription unit of an operon encompassing the IAA synthesis genes. These genes, designatediaaMandiaaH, reside on a 78-MDa native plasmid. The sequence analysis indicates that the operon contains two open reading frames of 562 (iaaM) and 460 amino acids (iaaH), corresponding to proteins with molecular weight of 62,473 and 49,300 daltons, respectively. When cloned and expressed in minicell-producingE. coli, this sequence encodes production of two proteins, 49 and 62 kDa. Both genes show moderate to high homology to IAA synthesis genes from other plant-associated bacteria. Analysis by primer extension of the transcription initiation site located it 63 bases upstream of the translation initiation codon.

Molecular cloning of the murine adenosine deaminase gene from a genetically enriched source: identification and characterization of the promoter region

1986 ◽  
Vol 6 (12) ◽  
pp. 4458-4466
Author(s):  
D E Ingolia ◽  
M R Al-Ubaidi ◽  
C Y Yeung ◽  
H A Bigo ◽  
D Wright ◽  
...  
Keyword(s):  
Adenosine Deaminase ◽  
Structural Gene ◽  
Transcription Initiation ◽  
Genomic Library ◽  
Gene Promoter ◽  
Mouse Cell ◽  
Gene Promoters ◽  
Sp1 Transcription Factor ◽  
Base Pair Fragment ◽  
Functional Analyses

A genomic library was prepared with DNA from a genetically enriched mouse cell line in which amplified copies of the adenosine deaminase (ADA) gene account for over 5% of the genome. Overlapping cosmid clones encompassing the entire ADA structural gene were isolated from this genomic library and used for subsequent structural and functional analyses. Nuclease protection and primer extension analyses served to identify the location of multiple transcription initiation sites at the 5' end of the structural gene. Promoter activity was found by functional analyses to reside within a 240-base-pair fragment which contains the transcription initiation sites. Sequences upstream of the transcription initiation sites are very G + C rich (77%) and include a 22 nucleotide stretch of deoxyguanylate residues and two potential Sp1 transcription factor-binding sites. Comparison of the mouse and human ADA gene promoters revealed the presence of several regions that are highly conserved with regard to both sequence content and location and may represent genetic elements which are involved in ADA gene expression.

scholarly journals Stimulation of sea urchin H2B histone gene transcription by a chromatin-associated protein fraction depends on gene sequences downstream of the transcription start site.

1985 ◽  
Vol 5 (10) ◽  
pp. 2764-2769 ◽  
Author(s):  
J Mous ◽  
H Stunnenberg ◽  
O Georgiev ◽  
M L Birnstiel
Keyword(s):  
Sea Urchin ◽  
Protein Fraction ◽  
Transcription Initiation ◽  
Initiation Site ◽  
Histone Gene ◽  
Xenopus Laevis Oocyte ◽  
Chromosomal Protein ◽  
Base Pairs ◽  
Sequence Element ◽  
Associated Proteins

We isolated a chromosomal protein fraction derived from chromatin of sea urchin embryos which specifically stimulated the expression of the histone H2B gene by a factor of 5- to 10-fold when the complete sea urchin histone gene repeat h22 was injected in Xenopus laevis oocyte nuclei. Gene manipulation experiments revealed the existence of two different target sites in the H2B gene which appear to mediate the response to injection of the stimulatory sea urchin chromatin-associated proteins; both are located downstream of the transcription initiation site. The first sequence element which is shown to be implicated is within, or at least includes, the H2B 5' untranslated leader sequence between nucleotides 11 and 76. The second element resides within an H2B DNA segment located near the 3' end of the gene, extending from 90 base pairs upstream of the mRNA 3' terminus to 140 base pairs in the spacer sequences downstream.

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 Genetic evidence for promoter competition in Saccharomyces cerevisiae.

1988 ◽  
Vol 8 (11) ◽  
pp. 4608-4615 ◽  
Author(s):  
J E Hirschman ◽  
K J Durbin ◽  
F Winston
Keyword(s):  
Transcription Initiation ◽  
Initiation Site ◽  
Base Change ◽  
Wild Type ◽  
Base Pairs ◽  
Cis Acting ◽  
Initiation Of Transcription ◽  
In Trans ◽  
Tata Sequence ◽  
Transcription Signals

The his4-912 delta mutation is an insertion of the long terminal repeat (delta) of the yeast retrotransposon Ty into the HIS4 promoter region, such that the delta is 97 base pairs upstream of the HIS4 transcription initiation site. Strains carrying the his4-912 delta allele are His- at 23 degrees C; this phenotype can be reversed either by growth at 37 degrees C or by mutations in trans-acting SPT genes. Under conditions in which his4-912 delta confers a His- phenotype. HIS4 transcription initiates at the delta initiation site, rather than at the HIS4 initiation site, producing a longer, nonfunctional transcript. Under conditions in which the strain is His+, transcription initiates at the wild-type HIS4 initiation site. To understand how transcription is balanced between the delta and HIS4 promoters, we have selected for cis-acting suppressors of his4-912 delta. Two classes defined by six independent mutations restore synthesis of a functional HIS4 transcript. The first class is an A-to-G base change 1 base upstream of the proposed delta TATA sequence. These mutants do not synthesize the delta-initiated transcript; instead, they synthesize only the wild-type HIS4 transcript. The second class of mutations alters base pairs surrounding the functional HIS4 TATA sequence. The two strongest His+ mutants of this class synthesize the wild-type HIS4 transcript at levels consistent with their His+ phenotype. Surprisingly, these two mutants also have a reduced level of the delta-initiated transcript relative to the his4-912 delta parent. Analysis of these mutants indicates that the level of transcription from one promoter can affect the level of transcription from the other promoter and suggests that delta and HIS4 transcription signals compete for initiation of transcription from each site.

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 Molecular cloning of the murine adenosine deaminase gene from a genetically enriched source: identification and characterization of the promoter region.

1986 ◽  
Vol 6 (12) ◽  
pp. 4458-4466 ◽  
Author(s):  
D E Ingolia ◽  
M R Al-Ubaidi ◽  
C Y Yeung ◽  
H A Bigo ◽  
D Wright ◽  
...  
Keyword(s):  
Adenosine Deaminase ◽  
Structural Gene ◽  
Transcription Initiation ◽  
Genomic Library ◽  
Gene Promoter ◽  
Mouse Cell ◽  
Gene Promoters ◽  
Sp1 Transcription Factor ◽  
Base Pair Fragment ◽  
Functional Analyses

A genomic library was prepared with DNA from a genetically enriched mouse cell line in which amplified copies of the adenosine deaminase (ADA) gene account for over 5% of the genome. Overlapping cosmid clones encompassing the entire ADA structural gene were isolated from this genomic library and used for subsequent structural and functional analyses. Nuclease protection and primer extension analyses served to identify the location of multiple transcription initiation sites at the 5' end of the structural gene. Promoter activity was found by functional analyses to reside within a 240-base-pair fragment which contains the transcription initiation sites. Sequences upstream of the transcription initiation sites are very G + C rich (77%) and include a 22 nucleotide stretch of deoxyguanylate residues and two potential Sp1 transcription factor-binding sites. Comparison of the mouse and human ADA gene promoters revealed the presence of several regions that are highly conserved with regard to both sequence content and location and may represent genetic elements which are involved in ADA gene expression.

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 Structure and transcription of the Drosophila melanogaster vermilion gene and several mutant alleles

1990 ◽  
Vol 10 (4) ◽  
pp. 1423-1431
Author(s):  
L L Searles ◽  
R S Ruth ◽  
A M Pret ◽  
R A Fridell ◽  
A J Ali
Keyword(s):  
Drosophila Melanogaster ◽  
Transcription Initiation ◽  
Donor Site ◽  
Initiation Site ◽  
Splice Donor Site ◽  
Wild Type ◽  
Upstream Site ◽  
Reading Frame ◽  
Exon 1 ◽  
A Minor

The nucleotide sequence and intron-exon structure of the Drosophila melanogaster vermilion (v) gene have been determined. In addition, the sites of several mutations and the effects of these mutations on transcription have been examined. The major v mRNA is generated upon splicing six exons of lengths (5' to 3') 83, 161, 134, 607, 94, and 227 nucleotides (nt). A minor species of v mRNA is initiated at an upstream site and has a 5' exon of at least 152 nt which overlaps the region included in the 83-nt exon of the major v RNA. The three v mutations, v1, v2, and vk, which can be suppressed by mutations at suppressor of sable, su(s), are insertions of transposon 412 at the same position in exon 1, 36 nt downstream of the major transcription initiation site. Despite the 7.5-kilobase insertion in these v alleles, a reduced level of wild-type-sized mRNA accumulates in suppressed mutant strains. The structure and transcription of several unsuppressible v alleles have also been examined. The v36f mutation is a B104/roo insertion in intron 4 near the splice donor site. A mutant carrying this alteration accumulates a very low level of mRNA that is apparently polyadenylated at a site within the B104/roo transposon. The v48a mutation, which deletes approximately 200 nt of DNA, fuses portions of exons 3 and 4 without disruption of the translational reading frame. A smaller transcript accumulates at a wild-type level, and thus an altered, nonfunctional polypeptide is likely to be synthesized in strains carrying this mutation.(ABSTRACT TRUNCATED AT 250 WORDS)

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