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PubMed1990

Identification and nucleotide sequence of the gene encoding a surface antigen induced by vaccinia virus.

The gene encoding the specific antigen (S antigen) induced on the surface of vaccinia virus-infected cells was mapped on the viral genome by the construction of recombinant viruses. The S gene is located in the 1.5-kbp XbaI-AccII fragment between 11.1 and 12.6 kbp from the right end of the viral genome. Nucleotide sequencing of the fragment revealed an open reading frame (ORF) of 351 codons capable of producing a polypeptide with a molecular weight (MW) of 40,701 Da. Mapping of the S mRNA by primer extension indicates the presence of the transcription initiation site that precedes the translation initiation codon by 14 nucleotides. The region preceding the S mRNA start site is extremely A/T-rich and has sequence similarity with other early genes. The signal for termination of transcription of the early genes, TTTTTAT, is included near the end of the ORF. The deduced amino acid sequence contains a typical membrane signal characterized by a hydrophobic region in the N-terminus. An early polypeptide with a MW of 40 kDa, in agreement with the size predicted from the ORF, was synthesized by cell-free translation of the mRNA selected by hybridization to a plasmid containing the region of the ORF.

Amino Acid Sequence↗

PubMed1983

Overlap and cotranscription of the genes for the beta and epsilon subunits of tobacco chloroplast ATPase.

The nucleotide sequences of the genes for the beta and epsilon subunits of tobacco chloroplast ATPase have been determined. The coding regions for the beta and epsilon subunits contain 1494 bp (498 codons) and 399 bp (133 codons), respectively. The 3' end of the beta-coding region overlaps by one nucleotide with the 5' end of the epsilon-coding region. The overlapping termination and initiation codons are ATGA. The beta and epsilon genes are cotranscribed as a 2.7-kb polycistronic mRNA. The amount of the beta and epsilon mRNA in the chloroplast is about one-twentieth that of the LS mRNA.

Base Sequence↗

PubMed1982

Spontaneous changes in nucleotide sequence in proviruses of spleen necrosis virus, an avian retrovirus.

We determined the nucleotide sequence of about 1 kilobase of DNA 3' to the 5' long terminal repeat of three noninfectious ad one infectious proviral DNA clones of spleen necrosis virus, an avian retrovirus, to determine if the types of nucleic acid changes involved in retrovirus mutation shed light on special features of retrovirus replication. An open reading frame was found starting 411 base pairs from the end of the long terminal repeat. It contained sequences coding for the 36 amino acids at the amino terminus of the p30 of a related reticuloendotheliosis virus [Oroszlan, S., Barbacid, M., Copeland T., Aaronson, S. A. & Gilden, R. V. (1981) J. Virol. 39, 845-854]. Therefore, the open reading frame represents the 5' end of the gag gene. A mutation in one noninfectious provirus changed the initiation codon for the gag polypeptide; a mutation in another noninfectious provirus caused premature termination of gag polypeptide synthesis; and a nontandem duplication into gag resulting from a mistake in initial (+) strand DNA synthesis changed amino acids and the reading frame in a third noninfectious provirus. These mutations appear to be responsible for the lack of infectivity of these provirus clones and indicate a higher relative frequency of mutation in this region of the genome. In addition, all four clones have multiple other mutations. These mutations are mostly base pair substitutions and many are clustered for any one clone, reflecting certain special features of retrovirus replication.

Amino Acid Sequence↗

PubMed1979

Formation of ribosome-RNA initiation complexes with alfalfa mosaic virus RNA 4 and RNA 3.

RNA 4 of alfalfa mosaic virus (AMV) is a monocistronic messenger for the coat protein. We have determined the sequence of the 40 +/- 2 nucleotides in RNA 4 that were protected in the initiation complex formed with wheat germ 80 S ribosomes from digestion by T1 or pancreatic ribonucleases. The AUG coat protein initiation codon was near the middle of this protected region. We have found two ribosome-binding sites in RNA 3. The principal one, near the 5' end, is the initiation site for the major translation product, a 35,000 dalton protein. The second site binds ribosomes only weakly, at the beginning of the "silent" coat protein cistron, and is similar but not identical to the initiation site protection site is discussed.

Base Sequence↗

PubMed1995

Nucleotide sequence and expression of the genes for the alpha and beta subunits of phycocyanin in Cyanidium caldarium.

The nucleotide sequence of the plastid-encoded operon containing genes for the alpha (cpcA) and beta (cpcB) subunits of phycocyanin in the unicellular red alga Cyanidium caldarium is described. cpcB is located 5' to cpcA and the two genes are separated by a 102-bp spacer region. The transcription start site of cpcBA was mapped to 80 bp upstream of the ATG initiation codon of cpcB. Promoter-like elements similar to the -10 (TATAAT) and -35 (TTGACA) consensus promoters in bacteria were found 6 and 31 bp upstream of the transcription initiation site. Northern blotting revealed an abundant 1.3-kb cpcBA transcript in illuminated cells, but this transcript was undetectable in dark-grown cells. Expression levels of cpcBA in cells incubated with 10(-6) M heme in the dark were similar to those in cells illuminated for 24 h. Cells illuminated with 150 microM gabaculine (an inhibitor of delta-aminolevulinate synthesis) or 10 mM levulinic acid (an inhibitor of delta-aminolevulinate dehydrase) lacked detectable cpcBA transcripts. In cells illuminated with 200 microM N-methyl-mesoporphyrin IX (an inhibitor of ferrocheletase), inhibition of cpcBA expression and phycocyanin synthesis was similar. These results provide strong evidence that light induction of the cpcBA operon is dependent on synthesis of heme.

Amino Acid Sequence↗

PubMed1995

The product of the pleiotropic Escherichia coli gene csrA modulates glycogen biosynthesis via effects on mRNA stability.

The carbon storage regulator gene, csrA, modulates the expression of genes in the glycogen biosynthesis and gluconeogenesis pathways in Escherichia coli and has been cloned, mapped and sequenced (T. Romeo, M. Gong, M.Y. Liu, and A.M. Brun-Zinkernagel, J. Bacteriol. 175:4744-4755, 1993; T. Romeo and M. Gong, J. Bacteriol. 175:5740-5741, 1993). We have now conducted experiments that begin to elucidate a unique mechanism for csrA-mediated regulation. Steady-state levels of glgC transcripts, encoding ADP-glucose pyrophosphorylase, were elevated by up to sixfold in a csrA::kanR mutant and were less than 6.5% of wild-type levels in a strain containing pCSR10 (csrA+), as shown by S1 nuclease protection analysis. The rate of chemical decay of these transcripts after adding rifampin to cultures was dramatically reduced by the csrA::kanR mutation. Deletion studies of a glgC'-'lacZ translational fusion demonstrated that the region surrounding the initiation codon was important for csrA-mediated regulation and indicated that neither csrA-mediated regulation nor stationary phase induction of glgC expression originates at the level of transcript initiation. Cell-free (S-200) extracts containing the CsrA gene product potently and specifically inhibited the in vitro transcription-translation of glg genes. The deduced amino acid sequence of CsrA was found to contain the KH motif, which characterizes a subset of diverse RNA-binding proteins. The results indicate that CsrA accelerates net 5'-to-3' degradation of glg transcripts, potentially through selective RNA binding.

Amino Acid Sequence↗

PubMed1982

Regulation of HIS4-lacZ fusions in Saccharomyces cerevisiae.

The beginning of the Saccharomyces cerevisiae HIS4 gene has been fused to the structural gene for Escherichia coli beta-galactosidase. This construction, which contains HIS4 DNA from -732 to +30 relative to the translation initiation codon, has been integrated into the yeast genome at two chromosomal locations, HIS4 and URA3. At both locations, this 762-base-pair stretch of DNA is sufficient for initiating expression of beta-galactosidase activity in S. cerevisiae and confers upon this activity the regulatory response normally found for HIS4.

Base Sequence↗

PubMed1995

Translational regulation of murine complement factor B alternative transcripts by upstream AUG codons.

Factor B (Bf), a constituent of the alternative pathway of complement activation, is encoded by a single gene that is located within the MHC. In murine kidney and intestine, two Bf transcripts (Bf short and Bf long), generated from distinct transcriptional initiation sites, are expressed in approximately equal amounts. In the liver, > 95% of Bf mRNA is the short transcript. To ascertain the biologic consequences of this tissue-specific mRNA polymorphism, we quantitated the effect of structural differences between the two transcripts on net Bf protein synthesis. Cell-free translation of Bf mRNA in vitro revealed that the rate of translation of Bf short is about twice that of Bf long. The 5' extension of Bf long includes four short open reading frames upstream of the authentic translational initiation codons. Mutation of all four upstream AUGs generates a Bf long transcript with a translational rate about equal to that of Bf short. This effect was primarily accounted for by mutation of the second AUG from the 5' end. Similar studies of Bf expression in vivo showed an approximately twofold difference in translational rate between Bf long and the transcript mutated at the upstream AUGs. Because systemic and local inflammation in kidney alters the ratio of Bf long and short, net production of complement protein Bf in extrahepatic tissues is regulated by both transcriptional and translational control mechanisms.

Alternative Splicing↗

PubMed1999

Overexpression in Escherichia coli and characterization of the chloroplast triosephosphate isomerase from spinach.

An important Calvin cycle enzyme, chloroplast triosephosphate isomerase (cpTPI) from spinach, has been cloned and expressed in up to 15% of the total cell protein using the P(L) expression vector in Escherichia coli. An even higher level expression, up to 36% of the total protein, was achieved by replacing the nucleotide sequence between the ribosomal binding site and the initial codon, ATG, with an AT-rich sequence. Computer modeling revealed that the moderate change in the standard free energy (5'-DeltaG degrees ) of mRNA secondary structure in the translation initial region might be the major factor which led to the later high-level expression. The overexpressed spinach cpTPI was soluble and fully active and was able to be purified beyond 95% purity by DEAE-Sepharose and Sephadex G-75, and around 55 mg of purified enzymes was obtained from 1 liter of cultured bacteria. With d-glyceraldehyde 3-phosphate as substrate, K(m (D-3-P)) is 0. 68 mM, V(max (G-3-P)) is 3.16 x 10(4) micromol/min. mg, and K(cat (G-3-P)) is 4.51 x 10(3)/s; with dihydroxyacetone phosphate as substrate, the corresponding values are 7.27 mM, 1.04 x 10(3) micromol/min. mg, and 1.16 x 10(2)/s, respectively.

Base Sequence↗

PubMed1992

The yeast SUA7 gene encodes a homolog of human transcription factor TFIIB and is required for normal start site selection in vivo.

Mutations in the Saccharomyces cerevisiae SUA7 gene were isolated as suppressors of an aberrant ATG translation initiation codon in the leader region of the cyc1 gene. Molecular and genetic analysis of the cloned SUA7 gene demonstrated that SUA7 is a single copy, essential gene encoding a basic protein (calculated Mr of 38,142) that is homologous to human transcription factor TFIIB. Analysis of cyc1 transcripts from sua7 strains revealed that suppression is a consequence of diminished transcription initiation at the normal start sites in favor of initiation at downstream sites, including a major site between the aberrant and normal ATG start codons. A similar effect was found at the ADH1 locus, establishing that this effect is not cyc1 gene-specific. Thus, SUA7 encodes a yeast TFIIB homolog and functions in transcription start site selection.

Amino Acid Sequence↗

PubMed1994

Participation of initiation factors in the recruitment of mRNA to ribosomes.

The step of protein synthesis which is normally rate limiting, formation of the 48S initiation complex, is catalyzed by the group 4 initiation factors. Collectively they recognize the 7-methylguanosine-containing cap of mRNA, unwind mRNA secondary structure, and allow scanning for the initiation codon by the small ribosomal subunit. The activities of the eIF-4 polypeptides are modulated by phosphorylation. Recent studies shed new light on the mechanism of assembly of the 48S initiation complex and the effect of phosphorylation of one of the eIF-4 polypeptides, the cap-binding protein eIF-4E.

Animals↗

PubMed1994

Complete structure of the gene encoding an immunodominant antigen of Dirofilaria immitis and larva-specific synthesis of primary transcript.

The complete gene encoding an immunodominant antigen of Dirofilaria immitis was isolated from a Charomid 9-36 genomic DNA library. This genomic DNA clone termed 'Dg2' was characterized by restriction mapping, DNA sequencing of the 5' flanking region, the exon/intron boundaries and the polyadenylation addition site. The Dg2 with 4872 bp in length consisted of five exons interspersed with four introns. These exons reveal a single open reading frame followed by a long 3' non-coding region of 1383 bp. The open reading frame of 969 bp encodes a polypeptide of 322 amino acids with a molecular weight of 34,400. The ATG translation initiation codon starts 22 nucleotides downstream from the 5' end of the first exon. The polyadenylation signal sequence. AATAAA, is located at the 3' end of the last exon. The transcription initiation site was determined by primer extension technique. S1 nuclease mapping analysis demonstrated that the primary transcript derived from Dg2 is synthesized in microfilariae but not in male or female adult worms. The result suggests that the stage-specific expression of Dg2 is regulated at the level of primary transcript.

Amino Acid Sequence↗

PubMed1978

Intervening sequence of DNA identified in the structural portion of a mouse beta-globin gene.

The unusual electron microscopic appearance of a hybrid formed between 9S mouse beta-globin mRNA and its corresponding cloned gene segment is caused by at least one, and possibly two, intervening sequences of DNA that interrupt the mouse beta-globin gene. Such an interpretation is consistent with a paradoxical restriction site pattern previously noted in this gene and with the nucleotide sequence of that portion of the gene that spans both structural and intervening sequences. The large intervening sequence, approximately 550 base pairs in length, occurs in the structural globin sequence and immediately follows the beta-globin codon corresponding to amino acid 104. A smaller, putative intervening sequence is located close to the 5' end of the beta-globin-coding sequence but may reside beyond its initiation codon. The beta-globin gene thus appears to be encoded in two, and possibly three, discontinuous segments.

Animals↗

PubMed1987

The untranslated leader of nuclear COX4 gene of Saccharomyces cerevisiae contains an intron.

The nuclear gene for subunit IV of cytochrome oxidase (COX4) in Saccharomyces cerevisiae contains a 342 bp intron which is contained entirely within the 5' leader of the message. Splicing of the intron results in removal of several small open reading frames; subsequently, the COX4 AUG becomes the 5' proximal initiation codon. A strain with an rna2- mutation fails to splice mRNA efficiently at restrictive temperature and was used to map the intron splice junctions by RNase protection. Two major mRNA initiation sites were mapped by primer extension of synthetic oligodeoxynucleotides. The splice junctions and internal TACTAAC box conform to consensus sequences previously determined from other yeast introns. One gene for subunit V of cytochrome oxidase (COX5b) has also been shown to contain an intron. The significance of introns in two nuclear genes encoding subunits of cytochrome oxidase is discussed.

DNA, Fungal↗

PubMed1991

Recombinant human chromosomal proteins HMG-14 and HMG-17.

Vectors for expressing human chromosomal proteins HMG-14 and HMG-17 in bacterial cultures under the control of the temperature-inducible lambda PL promoter have been constructed. The open reading frames of the cDNAs have been amplified by the polymerase chain reaction (PCR), utilizing amplimers containing desired restriction sites, thereby facilitating precise location of the initiation codon downstream from a ribosomal binding site. Expression of the recombinant proteins does not significantly affect bacterial growth. The rate of synthesis of the recombinant proteins is maximal during the initial stages of induction and slows down appreciably with time. After an initial burst of protein synthesis, the level of the recombinant protein in the bacterial extracts remains constant at different times following induction. Methods for rapid extraction and purification of the recombinant proteins are described. The recombinant proteins are compared to the proteins isolated from eucaryotic cells by electrophoretic mobility, Western analysis and nucleosome core mobility-shift assays. The ability of the proteins to shift the mobility of the nucleosome cores, but not that of DNA, can be used as a functional assay for these HMG proteins. A source for large quantities of human chromosomal proteins HMG-14 and HMG-17 will facilitate studies on their structure, cellular function and mechanism of interaction with nucleosomes.

Base Sequence↗

PubMed1981

Nucleotide sequences and mapping of novel heterogenous 5'-termini of adenovirus 2 early region 4 mRNA.

The major 5'-termini of human adenovirus type 2 early gene block 4 mRNA were sequenced. Poly(A+) polyribosomal RNA was isolated from Ad2 early infected cells, the 5'-terminal m7GPPP removed and the 5'-OH of the penultimate 2'-0-methylated nucleotide labeled with [gamma-32P]ATP using polynucleotide kinase. Ad2 E4 mRNA was purified by hybridization to the Ad2 EcoRI-C fragment and was digested with RNase T1. The resulting oligonucleotides were resolved by two dimensional paper electrophoresis-homochromatography. Four major and 3-4 minor 5'-terminal sequences were identified and characterized. The sequence of the 5'-terminal structures of the major four termini are: (1) m7GpppUmU(m)UUACACUGp, (2) m7GpppUmU(m)UACACUGp, (3) m7GpppUmU(m)ACACUGp, and (4) m7Gppp(m6)AmC(m)ACUGp. These major 5'-terminal sequences were aligned with nucleotide 325, 326, 327, and 329 from the righthand end of the known Ad2 DNA sequence (1) in the region mapped as the 5'-terminus of E4 mRNA by electron microscopy (2,3) and S1 nuclease-gel (4) mapping. Two potential ribosomal binding sites and an initiator codon were found at 40 to 65 nucleotides and about 80 nucleotides, respectively, from these heterogenous 5'-termini. Ad2 E4 major mRNA species appear to be unique since mRNA molecules initiate at a pyrimidine, perhaps by RNA polymerase stuttering, or they are products of an unusual type of RNA processing.

Adenoviruses, Human↗

PubMed1988

Molecular cloning and nucleotide sequence of the gene for the ribosomal protein S11 from the archaebacterium Halobacterium marismortui.

The gene encoding ribosomal protein S11 (Escherichia coli S15 homologue) from Halobacterium marismortui was cloned employing two synthetic oligonucleotide mixtures, 23 and 32 bases in length, as hybridization probes. The nucleotide sequence of the gene and the adjacent 5'- and 3'-flanking regions (1300 base pairs) were then determined by the dideoxy chain termination method. Comparison of the nucleotide sequence of the H. marismortui S11 gene with that of the E. coli S15 gene (rpsO) showed that the 3'-end of the S11 gene can be aligned with the entire E. coli S15 gene, sharing 44% identical nucleotides. It has been found that the S11 gene has a higher G + C content (G + C = 65%) than that of the E. coli S15 gene (G + C = 53%). This increase in G + C content specifically shows up as a preference for G + C in the 3rd position of the codon. Upstream of the S11 gene, an archaebacterial promoter sequence (GGACTTTCA) and a putative ribosomal binding site (GCGGT) have been found, 88 and 15 (or 24) base pairs from the initiation codon of the gene. In addition, an open reading frame could be identified immediately after the stop codon for the S11 gene. Northern blotting analysis using the S11 coding region as probe has shown that the S11 gene is located on a 2.4-kilobase mRNA, suggesting that it is cotranscribed with other downstream gene(s).

Amino Acid Sequence↗

PubMed1993

Transcriptional characterization of an alpha-zein gene cluster in maize.

A cluster of five alpha-zein subfamily 4 (alpha-zein SF4) genes are present in a 56 kb region of the maize W22 genome. Two types of alpha-zein SF4 genes are in the cluster. One of the genes, termed a type 1 (T1) alpha-zein SF4 gene, contains no early in-frame stop codons. Four of the genes, termed type 2 (T2) alpha-zein SF4 genes, contain one or two early in-frame stop codons. The base sequence of the T1 alpha-zein SF4 gene is similar (> 90%) to the sequences of any of the four T2 alpha-zein SF4 genes. However, their sequences differ markedly at distances greater than -875 bp upstream from the translation initiation codon of the alpha-zein coding region. This region of dissimilarity is well inside the functional 5'-flanking region for the genes since a 1.8 kb transcript is initiated in this region and the sequences of the T2 alpha-zein SF4 genes are similar in this region. Two sizes of mRNA transcripts, 1.8 kb and 0.9 kb, were detected in a gene specific manner for 4 of the 5 genes in this alpha-zein SF4 gene cluster. One of the T2 alpha-zein SF4 genes had only the 0.9 kb transcript. The RNA level for the 0.9 kb transcript of the T1 alpha-zein SF4 gene was 5- to 10-fold higher than the transcript levels of any of the T2 alpha-zein SF4 genes. In each case, the amount of the 0.9 kb transcript detected was at least 5-fold higher than the amount of the 1.8 kb transcript. A cDNA clone with a sequence identical to a T2 alpha-zein SF4 gene was isolated, providing the first direct evidence for the transcription of T2 alpha-zein genes containing early in-frame stop codon(s) in maize endosperm.

Base Sequence↗