Nucleotide Open Reading Frame Research Articles

Isolation of a Yeast Gene, SRH1, That Encodes a Homblogue of the 54K Subunit of Mammalian Signal Recognition Particle

A 1.7 kilobase HindIII fragment of Saccharomyces cerevisiae DNA was cloned by cross-hybridization with the Escherichia coli secY gene. The complete nucleotide sequence of the 2.6 kb fragment of the yeast genomic DNA containing the cross-hybridizing HindIII fragment was determined. The sequence showed no apparent similarity with that of the E. coli secY gene with the exception of a completely matched sequence of 21 bp, but it contained a 1,623 nucleotide open reading frame coding for a protein of 541 amino acids with a calculated Mr of 59,600. The N-terminal portion of 303 residues of the predicted sequence was homologous to the cytosolic domain of the alpha-subunit of the signal recognition particle receptor (SR alpha), including consensus sequence elements for a GTP binding site, whereas the C-terminal portion of 238 residues had an unusual methionine-rich domain containing several repetitive sequences. An mRNA of 2.0 kb was detected on Northern blotting analysis. The predicted sequence was 48% identical with the reported sequences of the 54K subunit of the mammalian signal recognition particle (SRP54) (Romisch K. et al. (1989) Nature 340, 478-483; Bernstein, H.D. et al. (1989) Nature 340, 482-486). We designated this gene as SRH1 (SRP54 homologue). Gene disruption experiments showed that the SRH1 gene product is essential for cell growth.

Functional analysis of a 603 nucleotide open reading frame upstream of the polyhedrin gene of Autographa californica nuclear polyhedrosis virus

The sequence of the 2000 nucleotides immediately upstream of the polyhedrin gene of the Autographa californica multiple nucleocapsid nuclear polyhedrosis virus has been determined. Comparative analysis of the data identified a 603 nucleotide open reading frame (ORF) separated from the polyhedrin gene coding sequences by 156 nucleotides and in the opposite strand of DNA. Northern hybridization analysis of polyadenylated RNA from infected cells highlighted a 3.7 kb species produced maximally at 12 h post-infection, but not in the presence of cycloheximide. Preliminary nuclease S1 analysis of the 5' end of this RNA suggested that it initiated at a position very close to that of the polyhedrin mRNA start site. Deletion of a portion of the ORF 603 from viruses containing the normal polyhedrin gene and the lacZ gene in lieu of polyhedrin did not affect replication in cell culture or the production of beta-galactosidase protein. A virus which lacked the ORF 603 gene but produced polyhedrin had similar infectivity in Trichoplusia ni larvae compared to the wild-type virus. The chloramphenicol acetyltransferase (CAT) gene was also inserted in lieu of the ORF 603 in a virus containing the lacZ gene instead of the polyhedrin (Ac.CAT.lacZ). Analysis of CAT expression revealed that a maximum level was reached at 16 h p.i. and that transcription was initiated in Ac.CAT.lacZ at the same site as for the normal gene.

Genetic and structural characterization of the avirulence gene avrBs3 from Xanthomonas campestris pv. vesicatoria

The avirulence gene avrBs3 from Xanthomonas campestris pv. vesicatoria was cloned and found to be localized on a self-transmissable plasmid. Genetic analysis of an avrBs3 insertion mutation revealed that avrBs3 constitutes a single locus, specifying the resistant phenotype on pepper plants. Southern blot experiments showed that no DNA sequences homologous to avrBs3 were present in other races of X. c. pv. vesicatoria, which are unable to induce a hypersensitive reaction on ECW-30R. However, the DNA of several different pathovars of X. campestris hybridized to the avrBs3 probe. A deletion analysis defined a region of 3.6-3.7 kb essential for avrBs3 activity. The nucleotide sequence of this region was determined. A 3561 nucleotide open reading frame (ORF1), encoding a 125,000 dalton protein, was found in the 3.7 kb region that was sufficient for avrBs3 activity. A second long ORF (2351 nucleotides) was identified on the other strand. A remarkable feature of both ORFs is the presence of 17 direct repeats of 102 bp which share 91%-100% homology with each other.

Molecular Cloning Sequence and Distribution of Rat Calspermin, a High Affinity Calmodulin-binding Protein

Calspermin is a heat-stable, acidic calmodulin-binding protein predominantly found in mammalian testis. The cDNA representing the rat form of this protein has been cloned from a rat testis lambda gt11 library. Sequence analysis of two overlapping clones revealed a 232-nucleotide 5'-nontranslated region, 510 nucleotides of open reading frame, a 148-nucleotide 3'-untranslated region, and a poly(A) tail. Authenticity of the clones was confirmed by comparison of a portion of the deduced amino acid sequence with the sequence of a tryptic peptide obtained from the rat testis protein. The lambda gt11 fusion protein was recognized by affinity purified antibodies to pig testis calspermin and bound 125I-calmodulin in a Ca2+-dependent manner. Calspermin cDNA encodes a 169-residue protein with a calculated Mr of 18,735. The putative calmodulin-binding domain is very close to the amino terminus of the protein. This region shows 46% identity with the calmodulin-binding region of rat brain Ca2+/calmodulin-dependent protein kinase II and 32% identity with the equivalent region of chicken smooth muscle myosin light chain kinase. The 5'-nontranslated region reveals significant homology with a portion of the catalytic region of the calmodulin-dependent protein kinase family. Calspermin contains a stretch of 17 contiguous glutamic acid residues in the central region of the molecule. Computer analysis predicts calspermin to be 81% alpha-helix and 14% random coil. Analysis of genomic DNA indicates calspermin to be the product of a unique gene. Northern blot analysis of rat testis RNA reveals a 1.1-kilobase mRNA. This RNA is restricted to testis among several rat tissues examined and could not be identified in total RNA isolated from testes of other mammals. Analysis of cells isolated from rat testis reveals calspermin mRNA to be predominantly expressed in postmeiotic cells indicating that it may be specific to haploid cells.

Identification of a cDNA clone that contains the complete coding sequence for a 140-kD rat NCAM polypeptide.

Neural cell adhesion molecules (NCAMs) are cell surface glycoproteins that appear to mediate cell-cell adhesion. In vertebrates NCAMs exist in at least three different polypeptide forms of apparent molecular masses 180, 140, and 120 kD. The 180- and 140-kD forms span the plasma membrane whereas the 120-kD form lacks a transmembrane region. In this study, we report the isolation of NCAM clones from an adult rat brain cDNA library. Sequence analysis indicated that the longest isolate, pR18, contains a 2,574 nucleotide open reading frame flanked by 208 bases of 5' and 409 bases of 3' untranslated sequence. The predicted polypeptide encoded by clone pR18 contains a single membrane-spanning region and a small cytoplasmic domain (120 amino acids), suggesting that it codes for a full-length 140-kD NCAM form. In Northern analysis, probes derived from 5' sequences of pR18, which presumably code for extracellular portions of the molecule hybridized to five discrete mRNA size classes (7.4, 6.7, 5.2, 4.3, and 2.9 kb) in adult rat brain but not to liver or muscle RNA. However, the 5.2- and 2.9-kb mRNA size classes did not hybridize to either a large restriction fragment or three oligonucleotides derived from the putative transmembrane coding region and regions that lie 3' to it. The 3' probes did hybridize to the 7.4-, 6.7-, and 4.3-kb message size classes. These combined results indicate that clone pR18 is derived from either the 7.4-, 6.7-, or 4.3-kb adult rat brain RNA size class. Comparison with chicken and mouse NCAM cDNA sequences suggests that pR18 represents the amino acid coding region of the 6.7- or 4.3-kb mRNA. The isolation of pR18, the first cDNA that contains the complete coding sequence of an NCAM polypeptide, unambiguously demonstrates the predicted linear amino acid sequence of this probable rat 140-kD polypeptide. This cDNA also contains a 30-base pair segment not found in NCAM cDNAs isolated from other species. The significance of this segment and other structural features of the 140-kD form of NCAM can now be studied.

Structure of the nucleocapsid protein gene of sonchus yellow net virus

The structure of the gene adjacent to the “leader RNA” gene of sonchus yellow net virus (SYNV), a plant rhabdovirus, was deduced by dideoxyribonucleotide sequence analysis of SYNV genomic (g) RNA and a series of plasmids constructed from SYNV gRNA or polyadenylated [poly(A) +] RNA from SYNV-infected plants. Evidence that this gene encodes the nucleocapsid (N) protein was obtained by reaction of SYNV N protein with polyclonal antibodies raised against recombinant proteins derived from the cloned gene. Experiments in which defined oligodeoxyribonucleotides were used to initiate reverse transcription of poly(A) + RNA from SYNV-infected tobacco revealed that the N protein messenger (m) RNA gene begins at position 147 from the 3′ end of the SYNV genome. Inspection of the sequence shows that this mRNA has a 56 nucleotide (NT) untranslated region followed by a 1425 NT open reading frame that is terminated by tandem UAA stop codons at positions 1628 to 1633 relative to the 3′ end of SYNV gRNA. Little direct sequence homology is evident between the 475 amino acid polypeptide predicted from the SYNV sequence and the nucleocapsid (N) proteins deduced from nucleotide sequences of the Indiana and New Jersey serotypes of vesicular stomatitis virus (VSV) and rabies virus. However, a short region of possible importance contains a small group of chemically similar amino acids common to all four N proteins.

CDNA sequence and tissue distribution of the mRNA for bovine and murine p11, the S100-related light chain of the protein-tyrosine kinase substrate p36 (calpactin I).

We have isolated and sequenced cDNA clones of bovine and murine p11 mRNAs. The nonpolyadenylated mRNAs are predicted to be 614 and 600 nucleotides, respectively. The p11 mRNAs both contain a 291 nucleotide open reading frame, preceded by a 5'-untranslated region of 73 nucleotides in bovine p11 mRNA and of 68 nucleotides in murine p11 mRNA. The deduced bovine p11 amino acid sequence is identical to the previously published partial bovine and complete porcine p11 protein sequence except for an additional COOH-terminal lysine residue. The bovine and murine p11 proteins are 92% homologous, whereas at the nucleotide level the conservation is 89% in the coding region and 75% in the 3'-untranslated region. Southern analysis of murine genomic DNA detected a single p11 gene, less than 10 kilobase pairs in size, containing as many as three introns. The p11 gene has been assigned to mouse chromosome 3 by analysis of interspecific hybrid cell panels and recombinant inbred mouse strains. The p11 gene is closely linked to the Xmmv-65 endogenous leukemia virus env gene and the guanylate binding protein-1 gene. Northern analyses of RNAs from mouse tissues and cell lines indicated that p11 mRNA levels vary widely. They are very low in liver, heart, and testes, moderate in brain, spleen, and thymus, and high in kidney, intestine, and lung. Analysis of the same RNA samples for p36 mRNA levels showed that expression of p11 and p36 mRNAs is not always coordinated. Brain and the mouse embryonal carcinoma cell line F9 contain moderate to high levels of p11 mRNA with very low levels of p36 mRNA. Sequence homology between p11 and the S100 proteins, and the serum-induced 2A9 gene product, as well as possible functions of p11 are discussed.

Structure and assembly of turnip crinkle virus: IV. Analysis of the coat protein gene and implications of the subunit primary structure

The structure of the turnip crinkle virus (TCV) coat protein and coat protein gene has been examined by cDNA cloning, nucleotide sequencing and high-resolution mRNA mapping. We have cloned a 1450-nucleotide cDNA fragment, representing the 3′ end of the TCV genome, using genomic RNA polyadenylated in vitro as the reverse transcriptional template. Nucleic acid sequence analysis reveals the presence of a 1053 nucleotide open reading frame capable of encoding a protein of 38,131 M r, identified as the coat protein subunit. The 1446 base subgenomic mRNA for the coat protein, mapped using high-resolution primer extension techniques, contains a 137 nucleotide leader sequence upstream from the initiation codon. We have characterized a second subgenomic RNA of approximately 1700 bases, roughly 250 nucleotides longer than the 1446 base species in the 5′ direction. No TCV-related RNAs are polyadenylated in vivo. The derived amino acid sequence of the TCV coat protein has been built into the 3.2 Å resolution electron density map of TCV reported in paper I of this series. We describe here some of the important features of the structure. Alignment of the three-dimensional structures of tomato bushy stunt virus and southern bean mosaic virus shows significant sequence relationships in the arms and S domains, although the conserved residues do not appear to have any special role in stabilizing the β-barrel fold or in mediating subunit interactions. The sequences of TCV and carnation mottle virus can be aligned. Comparisons among the four are discussed in terms of the organization of the S domain.

The Ace locus of Drosophila melanogaster: structural gene for acetylcholinesterase with an unusual 5' leader.

The Ace locus of Drosophila melanogaster has been mapped at the molecular level. cDNA clones from the locus have been isolated and their sequence determined, confirming that Ace forms the structural gene for acetylcholinesterase (AChE). The cDNAs have a 1950 nucleotide open reading frame from which the complete amino acid sequence of AChE has been deduced. The Drosophila enzyme is found to have extensive homology to the known sequence of Torpedo AChE. Ace cDNAs have an unusual structure with a long 5' leader and several short upstream open reading frames.

Complete nucleotide sequence of recD, the structural gene for the alpha subunit of Exonuclease V of Escherichia coli.

Intracellular amplification of the Escherichia coli RecB and RecC proteins does not result in an increase in Exonuclease V activity unless the level of a third protein, encoded between the recB and argA genes, is also amplified. Nucleotide sequence analysis of this region reveals a 1,824 nucleotide open reading frame which would encode a protein of 608 amino acids with a calculated molecular weight of 66,973. This is assumed to be the structural gene for the alpha subunit of Exonuclease V, recently designated recD. The proposed initiation codon of the recD gene overlaps the termination codon of the upstream recB gene by one nucleotide, suggesting that these genes may form an operon. The deduced amino acid sequence of the RecD protein contains a region which is homologous to highly conserved sequences in adenine nucleotide binding proteins.

TheILV1 gene in Saccharomyces cerevisiae: 5′ and 3′ end mapping of transcripts and their regulation

TheILV1 gene of Saccharomyces cerevisiae encodes threonine deaminase, the first enzyme in the biosynthesis of isoleucine. The 5′ and 3′ termini of theILV1 mRNA have been mapped by primer extension using AMV reverse transcriptase and S1 nuclease. A considerable heterogeneity was found at the 5′ end, which displays twenty different termini spanning a region of 78 nucleotides (from position −39 to −116 upstream of the ATG translation initiation codon). S1 nuclease mapping reveal one 3′ end 143 to 147 nucleotides downstream to the last codon in the 1,728 bp longILV1 coding region. The two most prevalent transcripts comprise about half of theILV1 mRNA and initiate at −39 and −40 at the sequence CAAG, not previously found to be a cap site in genes coding for amino acid biosynthesis enzymes. All the twenty 5′ termini are located within a 93 nucleotide open reading frame with its ATG at the symmetry point of a large imperfect tandem inverted repeat. The steady state level of theILV1 mRNA is derepressed in response to amino acid starvation (general amino acid control). The general control concensus sequence TGACT is found at positions −22, −132 and −307, the latter two being located upstream to the mRNA initiation sites. The relative abundance of mRNA molecules with different 5′ termini in cells derepressed to various extent was very similar. Three TATA like sequences and a CAAT box can be recognized upstream to the transcribed region. No intervening sequences were found to be presen in theILV1 transcription unit indicating that the entire 1,728 bp open reading frame is translated.

Nucleotide sequence of the heat shock regulatory gene of E. coli suggests its protein product may be a transcription factor

We have sequenced a cloned segment of E. coli chromosomal DNA that includes the heat shock regulatory gene htpR. This segment contains an 852 nucleotide open reading frame bounded by transcriptional and translational signals. Both in vivo and in vitro the cloned segment produces a single protein that migrates in gels with the cellular protein (F33.4) implicated as the htpR product. Properties of a cloned fragment of the coding sequence truncated at the promoter-distal end are consistent with this assignment. The htpR gene product appears homologous to the sigma factor of RNA polymerase, and the two proteins are predicted to have similar secondary structure. In addition, two regions of the predicted htpR product resemble protein-DNA contact points conserved in known DNA-binding proteins.

Nucleotide sequence of the ribulosebisphosphate carboxylase gene from Rhodospirillum rubrum

The nucleotide sequence of a cloned DNA fragment encoding ribulose bisphosphate carboxylase from the purple non-sulfur bacterium Rhodospirillum rubrum has been determined. The deduced amino acid sequence of a 1398 nucleotide open reading frame exhibits weak overall homology to the sequences reported for analogous enzymes from cyanobacteria, algae and angiosperms. Thus knowledge of the sequence is useful in attempts to identify structural features of the enzyme which are essential to catalysis. The gene is flanked by nucleotide sequences similar to those implicated in the initiation of translation and termination of transcription in other bacteria.

Sequence for human argininosuccinate synthetase cDNA.

The nucleotide sequence for human argininosuccinate synthetase cDNA was determined by analysis of six clones isolated from a single experiment. The sequence covered 1623 nucleotides including 76 bases of poly(A) and contained a 1236 nucleotide open reading frame encoding a protein of 46,434 daltons. In one cDNA isolate, a cloning artifact or perhaps RNA polymerase error involving addition of an A in a region of six A's within the coding sequence was documented. Single base variations in the 3' untranslated region were examined in detail since detection of DNA polymorphisms in the cDNAs could imply over-expression of both alleles at the active locus in canavanine-resistant cells, i.e. a trans-acting mechanism for enzyme overproduction. However, the sequence from five cDNAs suggested some single base artifacts, and DNA polymorphism remains uncertain. The occurrence of three tandem arginine codons in the 5' untranslated region of the cDNA suggested the possibility of an interaction of arginyl-tRNA with mRNA to regulate RNA processing or half-life as a mechanism for arginine-mediated repression.