Nucleic acid and protein sequence databases

Nucleic acid and protein sequences contain a wealth of informationof interest to molecular biologists. The advent of molecularsequence databases provides a unique opportunity for the computeranalysis of all available sequences. Sequence databases servetwo main functions: (i) to facilitate comparisons with newlydetermined sequences, and (ii) to act as a source of data forthe generation and testing of hypotheses concerning molecularsequence organisation and evolution. The large amounts of sequencedata now becoming available require that algorithms for databasesearching be fast and efficient and considerable progress isbeing made in this area.     

Construction and expression of a synthetic wheat storage protein gene

A synthetic wheat high-molecular-weight (HMW) glutenin storage protein gene analog was constructed for expression in E. coli. This first synthetic HMW-glutenin gene and future modifications are intended to allow systematic dissection of the molecular basis of HMW-glutenin role in the visco-elastic properties critical for wheat product processing and utilization. The design of the gene included four features: different construction strategies for the separate assembly of major polypeptide domains, the inclusion of convenient restriction sites for modifications, use of a codon selection similar to E. coli highly expressed genes, and the ability to produce repetitive sequence domains of exact numbers of defined repeats. The complete synthetic HMW-glutenin construct was 1908 bp, and contained 32 identical copies of one of the HMW-glutenin repetitive domain motifs. The gene expressed the novel HMW-glutenin protein to relatively high levels in bacterial cultures and the protein exhibited the known anomalous behavior of HMW-glutenins in SDS-PAGE.     

Nucleic acid and protein clocks.

The use of pairwise comparisons of correctly aligned DNA and protein sequences for the measurement of time in historical biology remains a contentious matter. However, the limited success of some molecular evolutionary clocks provides a stimulus to attempt to improve their resolution by the judicious selection of sequences for ease of alignment, commonality of function, taxonomic breadth and appropriate rates of evolution. Existing algorithms for correcting observed distances for superimposed nucleotide substitutions or amino acid replacements appear adequate for the task, given the noise that results from the inherent variability of the process. Some possible approaches are illustrated through the use of gene and protein sequences of the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase: an enzyme that is demonstrably homologous from purple bacteria to flowering plants.     

Nucleotide sequence of a gene from chromosome 1D of wheat encoding a HMW-glutenin subunit.

A high molecular weight glutenin gene in hexaploid wheat has been isolated by cloning in bacteriophage lambda and characterized. The gene corresponds to polypeptide 12 encoded by chromosome 1D in the variety "Chinese Spring". The coding sequence predicted contains seven cysteine residues six of which flank a central repetitive region comprising more than 70% of the polypeptide. These findings are related to the role of high molecular weight subunits in the viscoelastic theory of gluten structure.     

Nucleic acid and protein synthesis and loss of vigour in germinating wheat embryos

A study has been made of the RNA and protein synthesising systems of wheat embryos isolated from seed lots having high viability but differing in vigour. The rate of RNA and protein synthesis in wheat embryos during the early hours of germination is related to the vigour of the seed lot. The imposition of a stress factor, in the nature of a sub-optimal germination temperature, during germination of isolated wheat embryos magnifies the differences in rates of protein and RNA synthesis between high and low vigour seed. Using cell-free protein synthesising systems it has been demonstrated that an important difference between high and low vigour embryos lies in the relative levels of messenger RNA in the embryo. High vigour embryos contain relatively higher levels of poly A⁺-RNA (i.e. potential mRNA species) than lower vigour embryos and furthermore the level of poly A⁺-RNA in high vigour embryos increases during early germination whilst in lower vigour embryos the level decreases. The difference in poly A⁺-RNA levels accounts, at least partially, for the differences in rates of protein synthesis observed between embryos from high and low vigour wheat seed during early germination at both optimal and sub-optimal germination temperatures.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - poly A⁺-RNA polyadenylated RNA - GM germination medium - PMS post-mitochondrial supernatant fraction     

Nucleic acid metabolism in cold-treated wheat embryos

The incorporation of ₃₂P into nucleic acid fractions separatedon a MAK column was compared for normally germinated and cold-treatedwheat embryos. ₃₂P accumulation in DNA fraction was decreasedby cold treatment, although that in the RNA fractions was slightlypromoted. The synthesis of the fraction, probably mRNA, elutedafter the peak of heavy rRNA was enhanced in cold-treated embryosand suppressed when the embryos were cold-treated in the presenceof 8-azaguanine, an inhibitor of vernalization. (Received May 2, 1975; )     

Antibody identification, chromosome map assignment, and sequence analysis of a Rab escort protein homolog in Drosophila1

Using a polyclonal antiserum a cDNA encoding a Rab escort protein (REP) homolog in Drosophila has been identified and sequenced. The gene encodes a 511 residue protein with a predicted molecular mass of 56855 Da. Antibody labeling demonstrates that Drosophila REP protein is present in the early embryo and that it is being apportioned uniformly throughout the embryo in a process likely to be linked to the syncytial nuclear divisions. In situ hybridization to polytene chromosomes reveals that the Drosophila REP gene is located in the 56E region on the second chromosome. Drosophila REP is the first invertebrate REP homolog to be identified and characterized.     

Nucleic acid sequence analysis software for microcomputers

It is clear that a computer-aided data control system is required for even small laboratories generating nucleic acid data. While the molecular biologist at many universities and large research institutions has access to mainframe computers and nucleic acid sequence analysis software, many find it more convenient to perform sequence analysis on microcomputers that are typically located within the investigator's laboratory and totally dedicated to sequence storage and analysis, in essence giving the investigator more personal control of analysis activities than is sometimes possible with shared mini- or mainframe computers. New programs are being written and released at an increasing rate to perform increasingly more complex and specialized analyses using small computer-based systems. This trend will undoubtedly continue, fueled by the need to manage the ever increasing quantity of sequence data.     

Rapid protein domain assignment from amino acid sequence using predicted secondary structure

The elucidation of the domain content of a given protein sequence in the absence of determined structure or significant sequence homology to known domains is an important problem in structural biology. Here we address how successfully the delineation of continuous domains can be accomplished in the absence of sequence homology using simple baseline methods, an existing prediction algorithm (Domain Guess by Size), and a newly developed method (DomSSEA). The study was undertaken with a view to measuring the usefulness of these prediction methods in terms of their application to fully automatic domain assignment. Thus, the sensitivity of each domain assignment method was measured by calculating the number of correctly assigned top scoring predictions. We have implemented a new continuous domain identification method using the alignment of predicted secondary structures of target sequences against observed secondary structures of chains with known domain boundaries as assigned by Class Architecture Topology Homology (CATH). Taking top predictions only, the success rate of the method in correctly assigning domain number to the representative chain set is 73.3%. The top prediction for domain number and location of domain boundaries was correct for 24% of the multidomain set (+/-20 residues). These results have been put into context in relation to the results obtained from the other prediction methods assessed.     

mRNA sequence and deduced amino acid sequence of the mumps virus small hydrophobic protein gene.

The mRNA of a putative small hydrophobic protein (SH) of mumps virus was identified in mumps virus-infected Vero cells, and its complete nucleotide sequence was determined by sequencing the genomic RNA and cDNA clones and partial sequencing of mRNA. The SH mRNA is 310 nucleotides long excluding the poly(A) and contains a single open reading frame encoding a protein of 57 amino acids with a calculated molecular weight of 6,719. The predicted protein is highly hydrophobic and contains a stretch of 25 hydrophobic amino acids near the amino terminus which could act as a membrane anchor region. There is no homology between the putative SH protein of mumps virus and the SH protein of simian virus 5, even though the SH genes are located in the same locus in the corresponding genome. One interesting observation is that the hydrophobic domain of simian virus 5 SH protein is at the carboxyl terminus, whereas that of mumps virus putative SH protein is near the amino terminus.     

Nucleic acid synthesis for investigations of gene functions.

The chemical syntheses of genes encoding human c-Ha-ras and T4 endonuclease V are described. These genes have been used for studies of mutagenesis by damaged bases and recognition of nucleic acids by proteins. The modes of interaction of photo-damaged DNA and cognate antibodies have also been investigated by biochemical and physicochemical approaches.