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PubMed1978

Nucleotide sequence of the O gene and of the origin of replication in bacteriophage lambda DNA.

The nucleotide sequence of the O gene in bacteriophage lambda DNA is presented. According to two possible initiator codons, the primary structure of the O protein deduced from the DNA sequence consists of 278 or 299 amino acid residues. Structure and function of the O protein--one of the two phage initiator proteins for lambda DNA replication--are discussed in the light of a secondary structure model for the O protein. The central part of the O gene contains a cluster of symmetrical sequences extending over 160 base pairs. The point mutation of the cis-dominant replication mutant ti12 is located in this region.

Amino Acid Sequence↗

PubMed1999

Molecular cloning, enhancement of expression efficiency and site-directed mutagenesis of rat epidermal cystatin A.

A rat cystatin A cDNA clone was isolated from a lambda ZAP library representing newborn rat skin mRNA by screening with a synthetic oligonucleotide designed from amino acid sequence 15-23 of the cysteine proteinase inhibitor. The obtained clone contained a partial coding region of the inhibitor, lacking the 5'-untranslated region and coding sequence for the NH(2)-terminal 13 residues. The amino acid sequence deduced from the base sequence, Glu14-Phe103, coincided with that determined at the amino acid level. To obtain the recombinant cystatin A protein, the DNA was fused with a synthetic linker encoding its missing N-terminal 17 residues and introduced into an expression vector, pMK2. In Escherichia coli, however, the expression level of the semi-synthetic gene was low, 0. 5 mg of the purified recombinant protein per 1 liter culture being produced. Changing of the codon usage of the N-terminal region in a pET-15b expression system led to an increase in the yield depending on the instability of the putative secondary structure around an initiation codon of the mRNA. The expressed cystatin A showed identical characteristics with the authentic form except for the absence of the N-terminal acetyl blocking group. Using the expression system, two kinds of point mutation, the conservative Val54 in the first loop QxVxG region being changed to Lys and Glu, were introduced, but there was almost no effect on the inhibitory activity toward papain. This suggests that the conserved Val in the reactive site is not restricted and that the hydrophobicity of the position is not essential for the activity of rat cystatin A.

Amino Acid Sequence↗

PubMed2005

Infectious pancreatic necrosis virus VP5 is dispensable for virulence and persistence.

Infectious pancreatic necrosis virus (IPNV) is the causative agent of infectious pancreatic necrosis (IPN) disease in salmonid fish. Recent studies have revealed variation in virulence between isolates of the Sp serotype, associated with certain residues of the structural protein VP2. The isolates are also highly heterogenic in the coding region of the nonstructural VP5 protein. To study the involvement of this protein in the pathogenesis of disease, we generated three recombinant VP5 mutant viruses using reverse genetics. The "wild-type" recombinant NVI15 (rNVI15) virus is virulent, having a premature stop codon at nucleotide position 427, putatively encoding a truncated 12-kDa VP5 protein, whereas rNVI15-15K virus encodes a 15-kDa protein. Recombinant rNVI15-deltaVP5 virus contains a mutation in the initiation codon of the VP5 gene that ablates the expression of VP5. Atlantic salmon postsmolts were challenged to study the virulence characteristics of the recovered viruses in vivo. The role of VP5 in persistent infection was investigated by challenging Atlantic salmon fry with the recovered viruses, as well as with the low-virulence field strain Sp103 and a naturally occurring VP5-deficient mutant of Sp103. The results show that VP5 is not required for viral replication in vivo, and its absence does not alter the virulence characteristics of the virus or the establishment of persistent IPNV infection.

Animals↗

PubMed1988

Structure of the rabbit fast-twitch skeletal muscle calsequestrin gene.

We have isolated two overlapping 17-kilobase (kb) genomic clones which, together, encode the rabbit fast-twitch skeletal muscle calsequestrin. The calsequestrin gene is 8.6 kb in length and consists of 11 exons. S1 nuclease protection and primer extension analyses position the transcription initiation site 191 nucleotides upstream of the translation initiation codon. The 5'-flanking sequence contains a CCAAT sequence and two CG-rich sequences but does not have an identifiable TATA box sequence in the anticipated position just upstream of the transcription initiation site. The sequence of the calsequestrin gene confirmed the sequence deduced from cDNA clones derived from neonatal muscle mRNA (Fliegel, L., Ohnishi, M., Carpenter, M. R., Khanna, V. K., Reithmeier, R. A. F., and MacLennan, D. H. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1167-1171). We could not detect any potential alternative splicing of the transcript in an analysis of this region of the gene that would account for the different sequence found at the carboxyl terminus of adult rabbit fast-twitch skeletal muscle calsequestrin.

Amino Acid Sequence↗

PubMed1988

Evidence that the 5'-untranslated leader of mRNA affects the requirement for wheat germ initiation factors 4A, 4F, and 4G.

The efficiency of translation of alfalfa mosaic virus (AMV) RNA 4, barley alpha-amylase (B alpha A) mRNA, and two chimeric mRNAs, AMV 4-B alpha A and B alpha A-AMV 4 (in which the 5' leader sequences of the two mRNAs were interchanged), was measured in an S30 extract from wheat germ and a fractionated system from wheat germ in which translation could be made dependent upon initiation factor (eIF) 3, 4A, 4F, or 4G. In the S30 system, AMV RNA 4 and the chimeric mRNA AMV 4-B alpha A are translated much more efficiently than B alpha A mRNA and the chimeric mRNA B alpha A-AMV 4. When the S30 system was supplemented with high amounts of purified eIF-3, eIF-4A, eIF-4F, and eIF-4G, B alpha A and B alpha A-AMV 4 mRNAs were translated as efficiently as AMV RNA 4 and AMV 4-B alpha A mRNA. These findings indicated that the mRNAs containing the B alpha A leader sequence required higher amounts of one or more of the initiation factors (eIF-3, eIF-4A, eIF-4F, and eIF-4G) for efficient translation. Determination of the amounts of the initiation factors required for translation in the fractionated system showed that AMV RNA 4 required 2-4-fold lower amounts of eIF-3, eIF-4A, eIF-4F, and eIF-4G than did B alpha A mRNA. Replacement of the B alpha A leader sequence with that of AMV RNA 4 decreased the amounts of eIF-4A, eIF-4G, and eIF-3 required, but did not affect the amount of eIF-4F required. Replacement of the AMV RNA 4 leader sequence with that of B alpha A mRNA increased the amounts of eIF-4F, eIF-4G, and eIF-3 required, but did not affect the amount of eIF-4A required. These data strongly suggest that the amounts of the factors required are affected not only by the 5' leader itself but also by interactions between the 5' leader and a region(s) of the mRNA 3' to the initiation codon.

Capsid↗

PubMed1992

Prostaglandin endoperoxide synthase gene structure: identification of the transcriptional start site and 5'-flanking regulatory sequences.

The gene for the murine prostaglandin endoperoxide (PGH) synthase (8, 11, 14-eicosatrienoate, hydrogen-donor:oxygen oxidoreductase, EC 1.14.99.1) has been cloned. The gene was isolated from a mouse NIH 3T3 cell genomic library and is contained in four overlapping lambda FIXII bacteriophage clones. The gene spans approximately 22 kb and consists of 11 exons. Primer extension and RNAse protection assays indicate that transcription of the gene begins at an initiation site 63 nucleotides 5' to the ATG translation initiation codon. Neither TATA or CAAT boxes are present immediately upstream of the transcriptional start site, but SP1 binding sites are present at positions -47 to -42 and -30 to -25, relative to the transcription initiation site. Examination of the 5'-end and 2400 bp of the 5'-flanking sequence of the gene revealed sequences with homology to several transcriptional regulatory sequences. Three putative AP-1 binding sites were found, two within the first exon and intron and another at position -2097 to -2090. The AP-1 site at position -2097 is adjacent to a sequence with similarity to a negative glucocorticoid regulatory element (nGRE) (position -2123 to -2009). The presence of AP sites by themselves, or in conjunction with an nGRE sequence, suggests a possible interplay between jun/fos regulatory proteins and the glucocorticoid receptor for positive and negative regulation of the PGH synthase gene. An unexpected finding was the presence at position -403 to -385 of a putative dioxin responsive element, a sequence found to be responsible for the induction of transcription of the cytochrome P450IA1 gene (CYPIA1) and other genes involved in detoxification/activation of polycyclic aromatic hydrocarbons.

Animals↗

PubMed1989

DNA sequence and comparative analyses of the equine herpesvirus type 1 immediate early gene.

The immediate early (IE) proteins of herpesviruses are important regulatory factors which control the expression of genes at the transcriptional level. We report the DNA sequence of the immediate early gene of the alphaherpesvirus equine herpesvirus type 1 (EHV-1). This sequence is shown to be extremely rich in guanine and cytosine, resulting in a highly biased codon usage. The IE gene region possesses 38 open reading frames (ORFs) greater than 300 bp in length, 11 of which have coding regions of at least 100 amino acids (aa) following potential translation initiator codons. The largest ORF consists of 1487 codons (4461 bp) starting with the first ATG and would encode a protein of MW 155,000. TATA and CCAAT sequences as well as several potential cis-acting elements lie upstream to the major ORF. The deduced amino acid sequence for the 155,000 protein has a high degree of homology to the herpes simplex virus type 1 (HSV-1) ICP4 protein and its varicella-zoster virus (VZV) homolog. The regions of the EHV-1 IE protein that are homologous with these proteins correspond to the previously determined pattern of homology between the HSV and VZV IE polypeptides. However, there are are a number of differences within these broadly defined regions. It is therefore expected that this comparative study will facilitate the identification of functionally important residues within the amino acid sequence of IE proteins.

Amino Acid Sequence↗

PubMed1997

Cloning and expression of the glutamate racemase gene of Bacillus pumilus.

A glutamate racemase gene (murI) was found in Bacillus pumilus cells and cloned into Escherichia coli WM335, a D-glutamate auxotroph, by means of a genetic complement method. MurI of B. pumilus encodes a 272-amino acid protein with an unusual initiation codon, TTG. The deduced amino acid sequence shows significant similarity with those of glutamate racemases from E. coli (ratio of identical residues, 28%), Pediococcus pentosaceus (44%), and Staphylococcus haemolyticus (49%). B. pumilus MurI was expressed as a fusion protein connected to the N-terminal 12 residues of beta-galactosidase; the fusion protein showed glutamate racemase activity, and resembled the enzyme of P. pentosaceus in physicochemical and enzymological properties.

Amino Acid Isomerases↗

PubMed1993

Molecular cloning and characterization of the mouse RB1 promoter.

We report the isolation and characterization of the mouse RB1 promoter and surrounding DNA sequences, and the identification of elements required for basal transcriptional activity. The mouse RB1 promoter, like the human homologue, has a high G + C content, constitutes a CpG island and is devoid of typical TATA and CAAT boxes. The first 235 base-pairs upstream of the translation initiation codon in the mouse promoter exhibit 80% sequence homology with the human sequence. This homology includes a region which contains putative binding sites for the transcription factors Sp1, ATF and E2F/DRTF1. Four major transcription initiation sites were identified downstream of this conserved region. Mutational analysis revealed that the Sp1 and ATF binding sites, but not the putative E2F/DRTF1 binding site, are critical for promoter activity. Complete disruption of the putative Sp1 and ATF sites abrogated transcription, whereas the introduction of point mutations, previously identified in the Sp1 and ATF sites in two low penetrance retinoblastoma families, reduced promoter activity in a cell type specific manner. Less reduction in activity occurred in retinoic acid induced differentiated P19 cells and NIH3T3 mouse fibroblasts than in undifferentiated embryonal carcinoma P19 cells. Activity of the RB1 promoter was found to be stimulated in retinoic-acid induced differentiated P19 cells compared to undifferentiated P19; this stimulation required intact Sp1 and ATF sites but not the putative E2F/DRTF1 binding site. Our results indicate that basal level of RB1 expression is governed by Sp1 and ATF.

Animals↗

PubMed1994

Cloning of two halohydrin hydrogen-halide-lyase genes of Corynebacterium sp. strain N-1074 and structural comparison of the genes and gene products.

We cloned the genes from Corynebacterium sp. strain N-1074, for two halohydrin hydrogen-halide-lyase isoenzyme (hheA and hheB), which are involved in the transformation of alpha, beta-halohydrin into the corresponding epoxide and the reverse reaction. The nucleotide sequences of 1057 base pairs including the hheA gene and of 1130 base pairs including the hheB gene were analyzed. The predicted amino acid sequence of the hheA gene product consisted of 244 residues, and the calculated molecular weight was 26,465. The hheB gene had two sets of potential ribosome binding site and a possible initiation codon. The predicted amino acid sequences of the gene products consisted of 235 and 227 residues, and the calculated molecular weights were 26,179 and 25,236, respectively. Site-directed mutagenesis experiments strongly suggested dual translational initiation in the hheB gene. Comparison of the predicted amino acid sequences for the hheA and hheB genes found significant homology only in the carboxyl terminal region. An analysis of the upstream regions of the both hhe genes suggested the presence of putative epoxide hydrolase genes, which might be involved in further degradation of epoxide compounds.

Amino Acid Sequence↗

PubMed1985

The effects of hybrid ribosome-binding-site variants on the expression of human interferon-beta in Escherichia coli.

Human beta-interferon (IFN-beta) has been expressed in Escherichia coli by using vectors that join the trp promoter and a hybrid ribosome-binding site (HRBS) to the mature IFN-beta coding sequence. Introduction of an NcoI site at the ATG initiation codon of IFN-beta by site-directed mutagenesis has facilitated the construction of a series of portable HRBSs, by utilizing oligodeoxynucleotide adapters. The spacing between the Shine-Dalgarno (S-D) sequence and the initiator ATG ranged from 7-13 bp. One spacer of 9 bp length was varied at a single position. IFN-beta expression by these HRBS variants, as analyzed by antiviral activity and SDS-PAGE, shows both nucleotide sequence and spacer length effects. In vitro transcription-translation experiments indicate that some single base changes in the HRBS significantly decrease the rate of translation of the IFN-beta mRNA.

Codon↗

PubMed1988

The 5'-upstream region of the yeast 25S rRNA gene contains a promoter element allowing expression in yeast and E. coli.

The 25S rRNA gene of Saccharomyces cerevisiae is preceded by a bona fide TATA sequence which allows the initiation of transcription--presumably by polymerase II--from the same strand as the 25S rRNA gene. When the promoter fragment is cloned in front of a lacZ gene equipped with an initiation codon but lacking a promoter, this element permits formation of beta-galactosidase both in yeast and E. coli. Using RNA from yeast transformed with the fusion plasmid, we mapped by primer elongation a single initiation site 63 bp downstream from the presumed TATA sequence, i.e. about 53 bp 5' of the 25S rRNA gene. A similar signal at about the same position was observed when RNA from untransformed wild-type yeast was used as a template for primer elongation. These results suggest that transcription from this polymerase II promoter-like element occurs in vivo. A regulatory function could not be assigned to this transcript. Its initiation is not significantly influenced by heme or carbon source, although two boxes of high homology with upstream activation sequences (UAS) mediating heme dependent expression of the iso-1-cytochrome c gene (CYC1) precede the promoter at the appropriate distance.

Amino Acid Sequence↗

PubMed1990

The gene encoding SMR1, a precursor-like polypeptide of the male rat submaxillary gland, has the same organization as the preprothyrotropin-releasing hormone gene.

SMR1 is a precursor-like polypeptide of the submaxillary glands of rats. Sequence analysis predicts that it could be processed by maturation enzymes to release a small peptide resembling the thyrotropin-releasing hormone. The SMR1 gene was isolated from a rat genomic library and sequenced. The SMR1 gene spans 4.7 kb and consists of three exons. The two introns occur a few nucleotides before the initiation codon in the 5' untranslated region, and a few nucleotides before the first predicted processing site, respectively. Such a structure is reminiscent of that of the preprothyrotropin-releasing hormone gene. The site of transcriptional initiation of the SMR1 gene was determined and 1.4 kb of 5'-flanking sequence was sequenced. The sequence analysis revealed the presence of alternating purine-pyrimidine tracts and of purine-rich sequences. In addition, some sequences which could be involved in the regulation of SMR1 gene expression were identified.

Amino Acid Sequence↗

PubMed1989

The translation start signal region of TEM beta-lactamase mRNA is responsible for heat shock-induced repression of amp gene expression in Escherichia coli.

pBR322 contains the amp gene encoding beta-lactamase. When Escherichia coli carrying this plasmid is exposed to heat shock, beta-lactamase synthesis is repressed transiently at the translational level. To identify the DNA element responsible for this translational repression, DNA segments containing the translation start region of the amp gene were excised from pAT153 and fused in frame with the lacZ reading frame in the open reading frame vector pORF1. These constructs were introduced into E. coli, and the effect of heat shock of the cells on the synthesis of beta-galactosidase starting from the amp start codon was examined. As is the case for pBR322-encoded synthesis of beta-lactamase, the synthesis of beta-galactosidase encoded by the fused genes also ceased transiently upon heat shock. It is concluded that the heat shock-induced repression of the amp gene occurs at the initiation step of translation. As far as the present study is concerned, the minimum DNA segment responsible for the repression is AT TGA AAA AGG AAG AGT ATG AG, which includes the Shine-Dalgarno sequence (AAGGA) and the initiation codon (ATG).

Base Sequence↗

PubMed1984

Expression from an internal AUG codon of herpes simplex thymidine kinase gene inserted in a retrovirus vector.

We identified structural features that affect the expression of an exogenous gene inserted into a retrovirus vector constructed by using spleen necrosis virus, an avian retrovirus. The thymidine kinase gene from herpes simplex virus type 1 containing deletions in the promoter and terminal sequences of the mRNA was inserted into spleen necrosis virus. We found that synthesis of thymidine kinase by the recovered virus was apparently initiated from internal AUG residues. At least in some cases, however, the level of expression depended on the number of AUGs and the nucleotide sequence around the AUGs that preceded the initiator codon of the thymidine kinase gene.

Codon↗

PubMed1996

The presence of a polymorphism at the translation initiation site of the vitamin D receptor gene is associated with low bone mineral density in postmenopausal Mexican-American women.

We examined the association of bone mineral density (BMD) with a polymorphism in the gene encoding the vitamin D receptor (VDR) that causes a change in the predicted protein sequence. The polymorphism results from a C-to-T transition and creates an initiation codon (ATG) three codons proximal to a downstream start site. The polymorphism can be defined by a restriction fragment length polymorphism (RFLP) using the restriction endonuclease FokI. The presence of a FokI site, designated f, allows protein translation to initiate from the first ATG. The allele lacking the site (designated F), initiates from a second ATG site. Thus, translation products from these alleles are predicted to differ by three amino acids with the f variant elongated. In a group of 100 postmenopausal Mexican-American Caucasian women, subjects with the ff genotype (15% of the study population) had a 12.8% lower BMD at the lumbar spine than FF subjects (37% of the population) (p = 0.01). Heterozygote (Ff) subjects (48% of the population) had an intermediate BMD. This association between BMD and genotype was not apparent at the femoral neck or forearm. Over a 2-year follow-up period, a decrease in BMD at the femoral neck was greater in ff compared with FF subjects (-4.7% vs. -0.5%, p = 0.005). This trend was not apparent at the lumbar spine or forearm. There were no differences between genotype groups in measurements of 25-hydroxyvitamin D (25(OH)D), calcitriol, parathyroid hormone (PTH), osteocalcin, or urinary pyridinolines. We conclude that the FokI polymorphism of the VDR gene correlates significantly with decreased BMD at the lumbar spine and with an increased rate of bone loss at the hip in ff subjects. We emphasize that these initial data should be interpreted with caution but that the utility of this polymorphism as a genetic marker to determine BMD and osteoporosis risk warrants further study in larger populations with subjects of diverse ethnic backgrounds.

Aged↗

PubMed1986

Complete nucleotide sequence of the topA gene encoding Escherichia coli DNA topoisomerase I.

The nucleotide sequence of a 4071 base-pair long segment containing the gene topA encoding Escherichia coli DNA topoisomerase I and its flanking regions has been determined. The gene encodes a total of 864 amino acids from the ATG start to a TAA termination codon, of which the first f-Met appears to be removed after translation; the calculated molecular weight of the translated protein is 97,413. Mapping of promoters by deletion of sequences upstream from the ATG initiation codon indicates the existence of at least two promoters that direct transcription into topA.

Amino Acid Sequence↗

PubMed1996

Cloning and characterization of the gene encoding pyruvate phosphate dikinase from Giardia duodenalis.

This paper reports the cloning and molecular characterization of the gene encoding pyruvate phosphate dikinase (PPDK) from Giardia. The ORF is 2652 nucleotide residues in length and not interrupted by introns. The gene appears to exist as a single copy in the genome and predicts a 97629 Da protein containing 884 amino acid residues. Comparison of the deduced Giardia PPDK sequence with those of homologous enzymes from other organisms revealed high sequence similarities and the presence of various conserved domains known to be essential for substrate binding and catalysis. Analysis of the ppdk gene and 19 other protein-coding genes from the protist revealed no typical TATA boxes, positioned at around -30, but the presence of two novel consensus sequence motifs in the 5' flanking regions. One is an AT-rich element immediately preceding the translation initiation codon and the other a 14-bp box centered at -30. These shared consensus sequence patterns present in the 5' flanking region of Giardia genes are suggested to play a role in the control of transcription initiation.

Animals↗