scholarly journals Structure of the mouse pore-forming protein (perforin) gene: analysis of transcription initiation site, 5' flanking sequence, and alternative splicing of 5' untranslated regions.

1991 ◽  
Vol 173 (4) ◽  
pp. 813-822 ◽  
Author(s):  
B S Youn ◽  
C C Liu ◽  
K K Kim ◽  
J D Young ◽  
M H Kwon ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Consensus Sequence ◽  
Initiation Site ◽  
Untranslated Regions ◽  
Type I ◽  
Type Ii ◽  
Sequence Motifs ◽  
Flanking Sequence ◽  
Inhibitory Protein ◽  
Related Sequence

We studied the 5' untranslated regions (UTRs) of the mouse lymphocyte pore-forming protein (PFP, perforin, and cytolysin). 5' UTRs were determined by primer extension analysis, sequencing PFP cDNA clone PFP-7, ribonuclease protection assays, and amplification of poly(A)+ RNA of cytolytic T lymphocyte using polymerase chain reaction (PCR). Two alternatively spliced 5' UTRs, designated type I and type II, of 222 and 115 bp, respectively, were found associated with PFP. Type II is identical to type I, except for being 107 bp shorter in the second exon. This deletion was generated by the use of alternative acceptor splice sites. The mouse PFP gene (Pfp) encodes three exons, is separated by two small introns, and spans a chromosomal region of approximately 7 kb. The first exon contains 79 bp of 5' UTR, the second exon contains 143 or 36 bp of 5' UTR (type I or type II UTR, respectively) plus the NH2-terminal region of the mouse PFP, and the third exon contains the rest of the COOH-terminal mouse PFP. The organization of the mouse Pfp is similar to that of the human gene. Moreover, the 5' flanking sequence of the mouse Pfp is highly homologous to that of the human Pfp. In contrast to the human sequence, the more immediate 5' flanking sequence of mouse Pfp contains two tandem "TATA" box-related elements and a GC box, but lacks a typical CAAT box-related sequence. Several other enhancer elements were found further upstream, including cAMP-, phorbol ester-, interferon-gamma-, and UV-responsive elements, and PU box-like and NFkB binding site-like elements. In addition, we found a nuclear inhibitory protein-like element, a transcriptional silencer, and a pair of purine-rich sequence motifs that were found in other T cell-specific genes, and three repeats of GGCCTG that may be a variation of a highly repetitious GCCCTG consensus sequence found in human Pfp.

scholarly journals Location and characterization of two widely separated glucocorticoid response elements in the phosphoenolpyruvate carboxykinase gene.

1988 ◽  
Vol 8 (1) ◽  
pp. 96-104 ◽  
Author(s):  
D D Petersen ◽  
M A Magnuson ◽  
D K Granner
Keyword(s):  
Transcription Initiation ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Consensus Sequence ◽  
Simian Virus 40 ◽  
Regulatory Elements ◽  
Simian Virus ◽  
Maximal Response ◽  
Flanking Sequence ◽  
Base Pairs ◽  
Chimeric Genes

Chimeric genes were constructed by fusion of various regions of the 5'-flanking sequence from the phosphoenolpyruvate carboxykinase (GTP) (PEPCK) gene to the chloramphenicol acetyltransferase-coding sequence and to simian virus 40 splice and polyadenylation sequences. These were used to demonstrate that two glucocorticoid regulatory elements (GREs) combine to confer glucocorticoid responsiveness upon the PEPCK gene in H4IIE hepatoma cells. Both elements, a distal one whose 5' boundary is located between -1264 and -1111 base pairs and a proximal one located between -468 and -420 base pairs relative to the transcription initiation site, act independently, in various positions and orientations, and upon the heterologous thymidine kinase promoter. Each element accounts for half of the maximal response of the chimeric genes. Therefore, two widely separated enhancerlike elements contribute equally to the response of the PEPCK gene to glucocorticoid hormones. Neither of the PEPCK GREs contains the TGTTCT consensus sequence associated with most other GREs.

scholarly journals Tailless keratins assemble into regular intermediate filaments in vitro

1990 ◽  
Vol 97 (2) ◽  
pp. 317-324
Author(s):  
M. Hatzfeld ◽  
K. Weber
Keyword(s):  
Intermediate Filaments ◽  
Consensus Sequence ◽  
In Vitro Mutagenesis ◽  
Type I ◽  
Type Ii ◽  
Tail Domain ◽  
Filament Formation ◽  
Wild Type ◽  
Keratin 8

To study the influence of the non alpha-helical tail domain of keratins in filament formation, we prepared a truncated keratin 8 mutant, K8/tailless. Using site-directed in vitro mutagenesis we introduced a stop codon in the position coding for amino acid number 417 of the K8/wild-type sequence, thereby deleting 86 amino acids of the non alpha-helical tail domain but leaving the consensus sequence at the end of the rod domain intact. Expression of the truncated keratin 8 in Escherichia coli allowed us to purify the protein by a two-step procedure. The filament-forming capacity of the truncated K8 with wild-type K18 and K19 was analyzed using in vitro reconstitution. The in vitro assembly studies with K8/tailless and K18 wild-type indicate that the C-terminal tail domain of a type II keratin, including the homologous subdomain H2, is not required for filament formation. Moreover, reconstitution experiments with K8/tailless and K19, a naturally occurring tailless keratin I, show that the tail domains of type I as well as type II keratins are not an essential requirement for in vitro filament formation. Our results suggest that in vitro filament elongation does not depend on interactions between head and tail domains, although the tail domain might have a role in stabilization of intermediate filaments arising from certain keratin pairs.

Location and characterization of two widely separated glucocorticoid response elements in the phosphoenolpyruvate carboxykinase gene

1988 ◽  
Vol 8 (1) ◽  
pp. 96-104
Author(s):  
D D Petersen ◽  
M A Magnuson ◽  
D K Granner
Keyword(s):  
Transcription Initiation ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Consensus Sequence ◽  
Simian Virus 40 ◽  
Regulatory Elements ◽  
Simian Virus ◽  
Maximal Response ◽  
Flanking Sequence ◽  
Base Pairs ◽  
Chimeric Genes

Chimeric genes were constructed by fusion of various regions of the 5'-flanking sequence from the phosphoenolpyruvate carboxykinase (GTP) (PEPCK) gene to the chloramphenicol acetyltransferase-coding sequence and to simian virus 40 splice and polyadenylation sequences. These were used to demonstrate that two glucocorticoid regulatory elements (GREs) combine to confer glucocorticoid responsiveness upon the PEPCK gene in H4IIE hepatoma cells. Both elements, a distal one whose 5' boundary is located between -1264 and -1111 base pairs and a proximal one located between -468 and -420 base pairs relative to the transcription initiation site, act independently, in various positions and orientations, and upon the heterologous thymidine kinase promoter. Each element accounts for half of the maximal response of the chimeric genes. Therefore, two widely separated enhancerlike elements contribute equally to the response of the PEPCK gene to glucocorticoid hormones. Neither of the PEPCK GREs contains the TGTTCT consensus sequence associated with most other GREs.

Expression of tissue-specific Ren-1 and Ren-2 genes of mice: comparative analysis of 5'-proximal flanking regions

1984 ◽  
Vol 4 (11) ◽  
pp. 2321-2331
Author(s):  
L J Field ◽  
W M Philbrick ◽  
P N Howles ◽  
D P Dickinson ◽  
R A McGowan ◽  
...  
Keyword(s):  
Structural Gene ◽  
Transcription Initiation ◽  
Submaxillary Gland ◽  
Inbred Strains ◽  
Initiation Site ◽  
Sequence Motifs ◽  
Specific Expression ◽  
Tissue Specific ◽  
Reading Frame ◽  
A Minor

All inbred strains of mice carry the Ren-1 structural gene, which encodes the renin-1 isozyme, the classical renin activity found in kidneys. In addition, some strains carry a second renin structural gene, Ren-2, which encodes the predominantly expressed submaxillary gland renin isozyme, renin-2. Ren-1 and Ren-2 exhibit markedly different patterns of tissue-specific expression. In an effort to understand the molecular basis for this differential expression, detailed analysis of the genomic sequences corresponding to the Ren-1 and Ren-2 genes, and the transcripts originating from these loci, was undertaken. Sequence analysis of regions proximal to the structural genes indicated the presence of eucaryotic consensus sequences for transcription. These sequence motifs were strongly conserved between Ren-1 and Ren-2. Approximately 150 bases upstream from the major transcription initiation site, significant differences between these genes were apparent, including the presence of a repetitive DNA element in the Ren-2 copy as well as other breaks in homology and sequence curiosities. Strong homology between Ren-1 and Ren-2 resumed at a point ca. 200 bases further upstream on Ren-1. S1 analysis of submaxillary gland and kidney RNA populations indicated that the majority of transcripts initiate at homologous positions on Ren-1 and Ren-2. On a per cell basis, the accumulation of Ren-1 transcripts in the kidney and Ren-2 transcripts in the submaxillary gland are probably equivalent. These results suggest that it is tissue-specific utilization of the homologous start sites that is critical to their differential patterns of expression. Models which can account for this observation are presented. Interestingly, we found a minor fraction of transcripts initiating 5' to the major transcription start site. These transcripts encoded an open reading frame which may add an additional 23 amino acids to the N-terminus of the renin precursor.

scholarly journals De novo computational identification of stress-related sequence motifs and microRNA target sites in untranslated regions of a plant translatome

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Prabhakaran Munusamy ◽  
Yevgen Zolotarov ◽  
Louis-Valentin Meteignier ◽  
Peter Moffett ◽  
Martina V. Strömvik
Keyword(s):  
De Novo ◽  
Untranslated Regions ◽  
Sequence Motifs ◽  
Microrna Target ◽  
Related Sequence ◽  
Target Sites ◽  
Computational Identification

scholarly journals M-CAT binding factor, a novel trans-acting factor governing muscle-specific transcription.

1990 ◽  
Vol 10 (8) ◽  
pp. 4271-4283 ◽  
Author(s):  
J H Mar ◽  
C P Ordahl
Keyword(s):  
Transcription Initiation ◽  
Mutational Analysis ◽  
Troponin T ◽  
Marker Gene ◽  
Initiation Site ◽  
Sequence Motifs ◽  
Promoter Element ◽  
Specific Expression ◽  
Binding Factor ◽  
Distal Promoter

The cardiac troponin T (cTNT) promoter contains a highly muscle specific distal promoter element capable of conferring muscle-specific transcription from a heterologous TATA box-transcription initiation site. Three sequence motifs within this distal promoter element are conserved in the promoter and regulatory regions of many sarcomeric protein genes. Mutational analysis demonstrated that homologies to two of these conserved motifs (CArG/CBAR and MEF 1) were not required for activity of cTNT promoter-marker gene constructs in transfected embryonic skeletal muscle cells. In contrast, disruption of either or both copies of the conserved M-CAT motif (5'-CATTCCT-3') inactivated the cTNT promoter in these cells. Both M-CAT motifs were protected from DNase I cleavage in solution footprint assays by an M-CAT binding factor (MCBF) present in nuclear extracts from embryonic muscle tissue. M-CAT mutations that inactivated the cTNT promoter also disrupted MCBF binding, indicating that MCBF may be a key trans-acting factor required for muscle-specific expression of the cTNT promoter. MCBF also bound to the M-CAT motif in the distal promoter region of the skeletal alpha-actin gene, suggesting that it may play a role in the regulation of this and perhaps other muscle genes that contain M-CAT motifs.

scholarly journals Isolation and characterization of the androgen-dependent mouse cysteine-rich secretory protein-3 (CRISP-3) gene

1995 ◽  
Vol 309 (3) ◽  
pp. 831-836 ◽  
Author(s):  
U Schwidetzky ◽  
B Haendler ◽  
W D Schleuning
Keyword(s):  
Salivary Gland ◽  
Transcription Initiation ◽  
Genomic Library ◽  
Consensus Sequence ◽  
Initiation Site ◽  
Secretory Protein ◽  
Specific Expression ◽  
Isolation And Characterization ◽  
Eukaryotic Genes ◽  
Responsive Element

The mRNA for cysteine-rich secretory protein-3 (CRISP-3) was originally identified in the mouse salivary gland as an androgen-dependent transcript, and is closely related to CRISP-1 and CRISP-2 which are abundantly expressed in the epididymis and testis respectively. Overlapping phage clones encompassing the entire length of the CRISP-3 gene were isolated from a lambda EMBL3 genomic library and analysed. DNA sequencing revealed that the gene consisted of eight exons ranging between 55 and 740 bp in size, and seven introns. All exon-intron junctions conformed to the GT/AG rule established for eukaryotic genes. The length of the introns was determined by PCR and was found to vary between 1.0 and 3.7 kb, indicating that the gene spans over 20 kb of the mouse genome. Primer extension allowed the mapping of the major transcription initiation site to an adenine located at the appropriate position downstream of a bona fide TATA box, in a region corresponding well to the eukaryotic consensus sequence. Over 800 bp of CRISP-3 promoter region were determined and two regions almost exactly matching the androgen-responsive element consensus RGWACANNNTGTWCY detected. In addition, sequences described in the Drosophila melanogaster Sgs-3 gene as being involved in its salivary gland-specific expression as well as two putative OTF- and GATA-binding elements were also found.

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 Transcription of the Rhodobacter sphaeroides cycA P1 Promoter by Alternate RNA Polymerase Holoenzymes

1998 ◽  
Vol 180 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Barbara J. MacGregor ◽  
Russell K. Karls ◽  
Timothy J. Donohue
Keyword(s):  
Heat Shock ◽  
Rna Polymerase ◽  
Rhodobacter Sphaeroides ◽  
Transcription Initiation ◽  
Consensus Sequence ◽  
Initiation Site ◽  
E Coli ◽  
Rna Polymerase Holoenzyme

ABSTRACT These experiments sought to identify what form of RNA polymerase transcribes the P1 promoter for the Rhodobacter sphaeroidescytochrome c 2 gene (cycA). In vitro, cycA P1 was recognized by an RNA polymerase holoenzyme fraction that transcribes several well-characterizedEscherichia coli heat shock (ς32) promoters. The in vivo effects of mutations flanking the transcription initiation site (+1) also suggested that cycA P1 was recognized by an RNA polymerase similar to E. coli Eς32. Function of cycA P1 was not altered by mutations more than 35 bp upstream of position +1 or by alterations downstream of −7. A point mutation at position −34 that is towards the E. coliEς32 −35 consensus sequence (G34T) increasedcycA P1 activity ∼20-fold, while several mutations that reduced or abolished promoter function changed highly conserved bases in presumed −10 or −35 elements. In addition, cycA P1 function was retained in mutant promoters with a spacer region as short as 14 nucleotides. When either wild-type or G34T promoters were incubated with reconstituted RNA polymerase holoenzymes,cycA P1 transcription was observed only with samples containing either a 37-kDa subunit that is a member of the heat shock sigma factor family (Eς37) or a 38-kDa subunit that also allows core RNA polymerase to recognize E. coli heat shock promoters (Eς38) (R. K. Karls, J. Brooks, P. Rossmeissl, J. Luedke, and T. J. Donohue, J. Bacteriol. 180:10–19, 1998).

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.

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