Journal of molecular and applied genetics最新文献

In order to study the structural organization and regulation of the expression of the nitrogenase gene cluster in Rhizobium leguminosarum PRE we selected relevant subfragments of the sym-plasmid from clone banks by homology with R. meliloti nif-genes. Site-directed Tn5 mutagenesis was applied to a nif DH-specific clone and subsequently the transposon insertions were transferred back into the wild-type rhizobial genome by homologous recombination. Phenotypic effects of Tn5 mutations in the region of the structural nif-genes were determined by measuring acetylene reduction in nodulated plants and by immunological analysis of bacteroid-specific proteins. The localization of Tn5 insertion sites was in accordance with observed consequences: two genotypically different Tn5-induced mutations within nif D caused repression of CI alpha and beta synthesis and a strong reduction of CII production, thus resulting in a Fix- phenotype. Expression of different cloned Rhizobium DNA inserts, bearing nif K, nif D, nif H, or nif DH, was achieved in Escherichia coli minicells dependent upon the presence of a strong upstream vector promoter sequence. Gene products were identified by immunoprecipitation with specific antisera. Endogenous rhizobial transcriptional start signals in one case (nif H) seemed to be recognized at a low rate by the E. coli system; in contrast, Rhizobium ribosome binding sites for all three structural nif-genes functioned normally in minicells. The approximate location of the coding regions for nif KDH genes was determined and found to be contiguous.

Phytohemagglutinin (PHA), the major lectin of the common bean Phaseolus vulgaris, is synthesized during the development of the seeds. In most cultivars PHA makes up 5-10% of the total seed protein, but certain cultivars do not contain PHA. In vivo labeling of a normal cultivar (Greensleeves) and a PHA-minus cultivar (Pinto 111) showed that PHA was not synthesized in the PHA-minus cultivar. To find out whether the lack of synthesis was due to the absence of mRNA for PHA, recombinant cDNA clones for PHA were obtained. Total poly(A)+ RNA was isolated from cotyledons of developing seeds of Greensleeves and used to direct cDNA synthesis. The double stranded cDNA was cloned in pUC8 and transformants of Escherichia coli screened with pPVL134, a recombinant plasmid which contains the complete coding sequence for a PHA-like protein. Two weakly hybridizing clones (pSC1 and pSC2) were selected. Hybrid selection experiments showed that these two clones selected mRNAs which could be translated into polypeptides identical in size to PHA and recognized by antibodies to PHA. The recombinant pPVL134 selected mRNA which translated into polypeptides which were slightly smaller than those of PHA, and poorly recognized by antibodies to PHA. The recombinant clones were used to demonstrate that the genes for PHA and for the PHA-like protein are under temporal control during seed development. The cultivar Pinto 111, which has no detectable PHA, also has greatly reduced levels of mRNA for PHA. However, the gene for the PHA-like protein encoded by pPVL134 is expressed to the same degree in the cultivars Greensleeves and Pinto 111.

The DNA sequence has been determined for a 3.8-kilobase region which encodes the mercury resistance (mer) operon of the IncFII plasmid NR1. The sequence reveals four open reading frames which could encode proteins of 12,391, 9,429, 14,965, and 58,781 daltons. On the basis of their sizes, amino acid compositions, hydropathicities, and estimated isoelectric points, the peptides encoded by these open reading frames correspond to the four previously described Hg-inducible proteins detected in minicells carrying mer+ plasmids. The NR1 mer locus is 63.4% GC overall, and the Hg(II) reductase protein sequence is 90% homologous to that of Tn501. The region encoding the merR (positive regulatory) function has three open reading frames. The smallest of these possible merR peptides (6,457 daltons) begins approximately 280 bp to the right of the adjacent IS1b and reads towards the structural genes of the mer operon. The next largest reading frame (13,139 daltons) in the merR region begins 37 bp to the left of the beginning of the smallest peptide and also reads towards the structural genes. The largest reading frame (15,907 daltons) in the merR region lies on the complementary strand and reads away from the structural genes towards IS1b. Although attempts to visualize the merR gene product were not successful, in vitro mutagenesis allows us to eliminate the largest reading frame as a merR candidate. We were also able to show that approximately 50% of the smallest detectable mer peptide (9,429 daltons) is located in the periplasm.

Normal adult ducks possess two main types of hemoglobin: a major species (80%), HbI (alpha I2 beta I2), and a minor species (20%), HbII (alpha II2 beta II2). We have cloned recombinant cDNAs for the duck globin mRNAs using the ribosubstitution floppy loop technique. We present here the sequence for a duck alpha globin mRNA closely related to the chicken alpha D globin mRNA. Our new sequence data also include the 5' noncoding regions of the duck alpha A, alpha D, and beta globin mRNAs. Analysis of the untranslated regions of these mRNAs reveals several conserved sequences which may be important in the regulation of gene expression.

Plants regenerated from tobacco hairy root callus cultures were fertilized by self-pollination, and healthy, morphologically normal R1 offspring were obtained. Three of these R1 seedlings were analyzed for the presence of T-DNA and synthesis of the T-DNA-specific compound agropine. All three R1 plants analyzed contained the same full-length T-DNA as the parental regenerant, while only two showed agropine synthesis.

We have developed a highly efficient system for producing hepatitis B virus surface antigen in cultured mammalian cells. This system utilizes a recombinant bovine papilloma virus in which the hepatitis surface antigen coding sequences are inserted into the 5' untranslated region of the mouse metallothionein-I gene. Mouse fibroblasts stably transformed with this molecule produce surface antigen at levels as high as 10 mg/L/24 h and can be maintained in continuous culture for up to 85 days.

A recombinant plasmid (pcPvNGS-01) containing sequences related to glutamine synthetase (GS) has been identified from a cDNA library constructed from poly (A)+ RNA isolated from root nodules of Phaseolus vulgaris L. The identification of this recombinant relied on the observations that: (a) the clone hybridized strongly to purified GS mRNA; (b) in hybrid-select translation experiments, the clone selected mRNA that produced a polypeptide identical in molecular weight to purified GS subunits which was immunoprecipitated with anti-GS-antiserum; and (c) the translated nucleotide sequence of the cloned cDNA was homologous to a partial amino acid sequence of higher plant GS. The cloned cDNA hybridized to poly (A)+ RNA of different mobilities from leaves, roots, and nodules of P. vulgaris. In RNA "dot" blots washed at different stringencies, differences were observed both in the relative amounts of GS mRNA in different tissues and in the strength of their hybridization to the cDNA probe. The cloned probe hybridized to several fragments of restricted P. vulgaris DNA but not to DNA from Rhizobium phaseoli. These results suggest that GS is coded for by a small multigene family showing organ-specific expression.

Misincorporation by avian myeloblastosis virus reverse transcriptase in the presence of a single deoxyribonucleoside triphosphate was studied using 32P-labeled DNA primers annealed to the appropriate template DNA, and polyacrylamide-urea gel electrophoresis to measure the extension of the primer chains. With most primer-template combinations, greater than 50% of the primers were extended by the addition of a single incorrect nucleotide onto the end of the primer chain. Unexpectedly, one primer-template combination was not extended in the presence of dCTP, although misincorporation occurred with the other deoxyribonucleoside triphosphates. In another case, terminal misincorporation of two rather than one dT residue was observed. The primer termini containing unpaired nucleotides were efficiently extended upon addition of the other three deoxyribonucleoside triphosphates, even in the case where the primer terminus contained two unpaired nucleotides. Misincorporation was confirmed by direct sequence analysis. These results indicate that the frequency of mutations following misincorporation by reverse transcriptase in the presence of a single deoxyribonucleoside triphosphate should be sufficiently high to allow detection of mutants by simple screening procedures. An analysis of the sequence of a mutant resulting from misincorporation at the M13mp2 AvaII site revealed that following introduction of the DNA into Escherichia coli cells, mismatch repair preceded replication.

Several alpha-amylase clones were identified by screening a group of 345 cDNA clones derived from poly(A)+RNA from gibberellic acid (GA)-treated barley aleurone layer cells using an alpha-amylase cDNA clone that we had characterized previously as the hybridization probe. The alpha-amylase clones showed differences in the distribution of restriction enzyme sites, in the extent of cross hybridization, and in nucleotide sequence. There are at least three types of alpha-amylase clones in our collection, and the alpha-amylase cDNA clone reported by Rogers and Milliman [J. Biol. Chem. (1983) 258: 8169-8174] is a fourth type. The accumulation of alpha-amylase mRNAs in aleurones as a function of time after GA addition and of hormone concentration in the incubation medium was studied using different alpha-amylase cDNA clone probes. These studies indicate a differential control of the expression of the alpha-amylase genes by GA.

We describe the use of a combined cloning/expression protocol to identify a gene encoding a Pseudorabies virus (PRV) glycoprotein. Prior to this study, the genome locations of PRV glycoproteins had not been described. We first identified PRV glycoproteins using antibodies directed against PRV virions. Using affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the PRV glycoproteins were separated and antibodies were made against them in rabbits. Using DNase digestion, PRV genomic DNA was fragmented into approximately 500-base-pair regions. These random fragments were inserted in an expression vector and the PRV DNA sequences were expressed as proteins fused to beta-galactosidase. The antibodies made in rabbits were then used as probes in a Western blot analysis to screen for the presence of PRV-specific glycoprotein sequences in these PRV-beta-galactosidase fusion proteins. Two expression plasmids were isolated that specified fusion proteins that reacted in the Western blot analysis with rabbit antibodies directed against a 74,000 molecular weight PRV glycoprotein. The PRV DNA sequences represented in the expression plasmids were mapped within a single BamHI fragment of PRV genomic DNA, but were not overlapping. PRV-beta-galactosidase fusion proteins produced by these two expression plasmids were used to inoculate rabbits. Antibodies produced against both fusion proteins recognized PRV-specific glycoproteins of 74,000 and 92,000 molecular weight. This protocol should have general application in localizing genes within large DNA virus genomes.