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The alpha/beta barrel enzyme phosphotriesterase from soil bacteria appears to have evolved the ability to hydrolyze the insecticide paraoxon at the diffusion limit in only a few decades. A newly-identified open reading frame from Escherichia coli may offer a clue to its origins.
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The complete sequence of a 36775 bp DNA segment located on the right arm of chromosome XV of Saccharomyces cerevisiae has been determined and analysed. The sequence encodes 26 open reading frames of at least 100 amino acids. Eight of these correspond to known genes, whereas 18 correspond to new genes.
Flax rust, Melampsora lini strain SP6, contains 11 double-stranded (ds) RNA molecules with a total length of about 25 kbp. The dsRNAs are inherited in three genetic units: the L unit comprising a single 5.2 kbp dsRNA and contained within a 40-nm virus-like particle, and the A and B units each consisting of five dsRNAs (A1-A5, and B1-B5) ranging in size from 1.2 to 2.7 kbp. This paper reports the isolation of a cDNA library representing 10 of the 11 dsRNAs. By nucleic-acid hybridization techniques it has been shown that all ten sequences are unique showing no detectable cross-hybridization with any other dsRNA present in the rust. A near full-length sequence of 1932 bp of the B3 dsRNA is reported and contains several open reading frames, the largest of which comprises most of the molecule.
The 3'-ends of the genomes (9538 bp) of two wild-type respiratory bovine coronavirus (RBCV) isolates LSU and OK were obtained by cDNA sequencing. In addition, the 3'-end of the genome (9545) of the wild-type enteric bovine coronavirus (EBCV) strain LY-138 was assembled from available sequences and by cDNA sequencing of unknown genomic regions. Comparative analyses of RBCV and EBCV nucleotide and deduced amino acid sequences revealed that RBCV-specific nucleotide and amino acid differences were disproportionally concentrated within the S gene and the genomic region between the S and E genes. Comparisons among virulent and avirulent BCV strains revealed that virulence-specific nucleotide and amino acid changes were located within the S and E genes, and the 32 kDa open reading frame.
The paper presents the results of analysis of the primary sequence of repetitive barley DNA called BRS1 (index number in Genbank BRS-1 U72261). The sequence encodes the open reading frame for 337 amino acids which has homology to retrotransposon-like element of Arabidopsis. There is a block consisting of 11 direct and 7 inverted repeats outside the open reading frame, the sequence ATACTAATGGCGCACC being the base of the repeats.
The purpose of this chapter is not to present the final or even correct model of TSH receptor structure and function. Rather, the current speculative model presented is used to open the door to a more broad view of the receptor problem and controversy as it has evolved today. Questions of how we define a receptor are clearly very much in flux and much more difficult than initially considered when a chemical approach is taken. Numerous binding components will be described and their relevance to the physiologic state will be debated. Some will be clearly erroneous in concept--yet the very debate and data will open new ideas and approaches other than repetitive membrane binding or response measurements. The remainder of this book will explore numerous other aspects of receptor structure, regulation and function. The reader may be disturbed by the complexity and extrapolations of data and the weakness of the models. The reader should, however, remember that the receptor is the key link of the cell to its environment. The complexities of this linkage are evident in our continued concern with knowledge of the mechanisms our bodily senses utilize. The controversy that will exist is evident in the arguments we have today over the agents in our environment which affect us and the mechanisms of these effects. It is hoped that this chapter and book will provide both the desire and some reference to follow and review the data in all receptor fields as they emerge in the next several years.
Natural selection for an increased translation efficiency has been proposed as the main determinant for the bias in codon usage observed in many genes of Saccharomyces cerevisiae. Recently, the efficiency of transcription of a large number of yeast genes has been determined, based on the cellular content of the respective mRNAs: this provides an additional dimension to the study of the multisep process of gene expression. Using a representative set of yeast genes with a known level of transcription, the relationship between transcriptional and translational steps was evaluated by a multiple linear regression model. This analysis demonstrated a positive correlation between the amount of transcript, given as the number of mRNA copies per cell for each individual gene, and indices evaluating the effects of translational selection on the corresponding codon usage pattern. This finding suggests a close association of the cellular mRNA content, regulated also at the transcriptional level, to its efficiency of translation, mediated by a fine-tuning of codon usage strategy. Moreover, multiple regression analysis demonstrated that the transcription level of a gene can be approximately predicted using indices of bias deriving from its nucleotide sequence. This allowed for an extensive investigation of uncharacterized regions of the complete genome sequence of S. cerevisiae, to detect new potential short protein coding genes that were not considered by previous searching procedures. Several small open reading frames exhibiting a statistically significant coding potential were thus identified as good candidates for functional analysis.
We have cloned and sequenced the 5' untranslated region of the transforming growth factor-beta 3 (TGF-beta 3) mRNA as well as the adjacent genomic sequence. S1 nuclease analysis identified a single transcription start site. We have thus determined that the 5' untranslated region is about 1.1 kb long and contains 11 open reading frames. In vitro translation of the TGF-beta 3 precursor coding sequence was markedly inhibited by the presence of the 5' untranslated region. Similarly, when the 5' untranslated region of TGF-beta 3 was introduced upstream of the coding sequence of chloramphenicol acetyltransferase, in vitro translation was inhibited. Furthermore, upon transfection into 293 cells, chloramphenicol acetyltransferase expression was inhibited by the 5' untranslated region of TGF-beta 3. The degree of translational inhibition was inversely proportional to the amount of transfected DNA. Mutation analysis implicated multiple segments of the 5' untranslated region as contributing to the inhibitory effect. Deletion of much of the 5'-most 640 nucleotides, including 8 of the 11 upstream ATGs, relieved much but not all of the inhibitory influence of the 5' untranslated region of TGF-beta 3 mRNA. The two upstream open reading frames closest to the initiator codon for the TGF-beta 3 coding sequence also decreased translational efficiency, since mutation of either ATG resulted in increased translation. Transfection results with T47-D cells, a cell line which expresses TGF-beta 3 mRNA, were similar to those obtained with the 293 cell line. Thus, TGF-beta 3 mRNA is a recent example of an expanding group of growth-related mRNAs in which the 5' untranslated region contains upstream open reading frames and other sequences which inhibit translation.
The sequence of 380 nucleotides upstream of the start codon of the open reading frame (ORF) of the coat protein of the carlavirus Helenium virus (HelVS) was determined from cloned cDNA. This portion of the viral RNA contained one complete ORF encoding a protein of 7 K which had homology with similar sized proteins from other members of the carla- and potexvirus groups. Sequence data was also obtained beyond the start codon of the 7 K ORF into what was presumed to be the 12 K protein gene of the previously identified triple gene block of carla- and potexviruses. Significant sequence similarity of 46.3% and 41.6% was evident at the amino acid level with the equivalent regions of the 12 K proteins of potato virus S, Andean strain (PVSA) and potato virus M (PVM), respectively, with 25.4% similarity detected with the equivalent region of potato virus X (PVX).
The usefulness of random amplified polymorphic DNA markers (RAPD) was assayed in an attempt to discriminate among species, strains and individuals within the genus Schistosoma. Depending on the species, 40-50 arbitrary decamer oligonucleotides were used as primers to amplify total DNA by the polymerase chain reaction (PCR). An important polymorphism was observed among 5 species, allowing a phylogenetic tree to be outlined. These differences can be used for rapid and accurate identification. A limited but easily detectable polymorphism was revealed among 3 strains of a single species (Schistosoma mansoni). Minor differences were observed among individuals of a single strain. A RAPD marker allows sexual discrimination between individuals from the terminal spined-egg species group. Although a limited number of strains have been examined, the results already indicate clearly that RAPD markers constitute a powerful tool for the analysis of genetic variability. This new tool will considerably extend the information available from morphology, isozyme and limited restriction fragment length polymorphism data and opens the way to genetic analysis of these species.
The nucleotide sequence of cDNA encoding human erythrocyte AMP deaminase has been determined by screening of human spleen cDNA library and by utilizing polymerase chain reaction (PCR) techniques. The 3.7 kb cDNA contains an open reading frame of 2301 bp which encodes 767 amino acids chain resulting in 89 kDa protein. The polyadenylation consensus signal (5'-AATAAA) located at 1212 bp 3' downstream from the stop codon. The homologies to human and rat muscle-specific AMP deaminases showed 64.1% and 65.2% identities, respectively, at the nucleotide level in the area of open reading frame, and 60.2% and 59.8% similarities at the deduced amino acid level.
An oligomer probe was designed to detect the presence of a putative phoB gene in the genome of the marine, phycoerythrin-containing cyanobacterium Synechococcus sp. WH7803. A 2.2 kb PstI fragment, identified using this probe, was cloned and the complete nucleotide sequence determined. The fragment contained two open reading frames encoding polypeptides which display all the sequence features expected of the response regulator and histidine protein kinase elements of a two component sensory system. Northern analysis confirmed that transcription of these genes was induced by phosphate limitation. On the basis of the sequence similarities and the regulation of their transcription by the availability of inorganic phosphate (Pi) these open reading frames were designated as phoB and phoR, respectively.
Two tandemly located flagellin genes, flaA and flaB, with 79% nucleotide sequence identity were identified in Aeromonas salmonicida A449. The fla genes are conserved in typical and atypical strains of A. salmonicida, and they display significant divergence at the nucleotide level from the fla genes of the motile species Aeromonas hydrophila and Aeromonas veronii biotype sobria. flaA and flaB encode unprocessed flagellins with predicted Mrs of 32,351 and 32,056, respectively. When cloned under the control of the Ptac promoter, flaB was highly expressed when induced in Escherichia coli DH5alpha, and the FlaB protein was detectable even in the uninduced state. In flaA clones containing intact upstream sequence, FlaA was barely detectable when uninduced and poorly expressed on induction. The A. salmonicida flagellins are antigenically cross-reactive with the A. hydrophila TF7 flagellin(s) and evolutionarily closely related to the flagellins of Pseudomonas aeruginosa and Vibrio anguillarum. Electron microscopy showed that A. salmonicida A449 expresses unsheathed polar flagella at an extremely low frequency under normal laboratory growth conditions, suggesting the presence of a full complement of genes whose products are required to make flagella; e.g., immediately downstream of flaA and flaB are open reading frames encoding FlaG and FlaH homologs.
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Plasmid clone gapB and lambda phage clone 4682, which contain fragments of Saccharomyces cerevisiae chromosome VI, were analysed. A 23 kb sequence was determined and ten open reading frames (ORFs) were revealed. Among them, five ORFs were identical to five yeast genes (SEC4, MSH4, SPB4, DEG1 and NIC96), two were identical to transposable elements (TYA and TYB), one (gapBorfF003) was highly homologous to a yeast expressed sequence tag, and another (4682orfF002) was predicted to be a nuclear protein. Sequence data have been submitted to DDBJ/EMBL/GenBank data library under Accession Number D44604 (clone gapB) and D44600 (clone 4682), respectively.
We have sequenced a region of 51 kb of the right arm from chromosome XV of Saccharomyces cerevisiae. The sequence contains 30 open reading frames (ORFs) of more than 100 amino acid residues. Thirteen new genes have been identified. Thirteen ORFs correspond to known yeast genes. One delta element and one tRNA gene were identified. Upstream of the RPO31 gene, encoding the largest subunit of RNA polymerase III, lies a Abf1p binding site. The nucleotide sequence data reported in this paper are available in the EMBL, GenBank and DDBJ nucleotide sequence databases under the Accession Number X90518.
During sequence analysis of the first intron of the human c-fms oncogene, we identified an open reading frame encoding the ribosomal protein L7 (RPL7). The presence of this sequence within intron 1 of the c-fms gene was confirmed by Southern blot hybridization and by sequence analysis of two independent cosmid clones (cos2-e and cos1-22) that span the human genomic c-fms locus. The RPL7 sequence was detected in a region of sequence overlapped by the cos2-e and cos1-22 cosmid clones but oriented opposite to the c-fms gene. We demonstrated that the sequence is identical to the full-length RPL7 cDNA sequence, but lacks any recognizable introns, has a 30-bp poly(A) tail, and is bracketed by two perfect direct repeats of 14 bp. We also showed that despite the fact that the 5' flanking region of the RPL7 sequence contains a potential TATA box upstream of an intact open reading frame, this pseudogene (RPL7P) is not actively transcribed.