Nitrogenase promoter-lacZ fusion studies of essential nitrogen fixation genes in Bradyrhizobium japonicum I110.

DNA fragments containing either the nifD or nifH promoter and 5' structural gene sequences from Bradyrhizobium japonicum I110 were fused in frame to the lacZ gene. Stable integration of these nif promoter-lacZ fusions by homologous double reciprocal crossover into a symbiotically nonessential region of the B. japonicum chromosome provided an easy assay for the effects of potential nif regulatory mutants. The level of beta-galactosidase activity expressed from these two nif promoter-lacZ fusions was assayed in bacteroids of B. japonicum I110 wild type and Fix mutants generated by transposon Tn5 mutagenesis and identified in the accompanying paper. No nif-positive regulatory mutants were identified from among an array of Fix- mutants in which Tn5 was inserted 9 kilobase pairs upstream of the nifDK operon and within the 18-kilobase-pair region separating the nifDK and nifH operons. This result indicates that there are no genes in these regions involved in the regulation of nitrogenase structural gene expression. Interestingly, the level of beta-galactosidase activity expressed from the nifH promoter was twice that expressed from the nifD promoter, suggesting that the normal cellular level of the nifH gene product in bacteroids is in a 2:1 ratio with the nifD gene product instead of in the 1:1 stoichiometry of the nitrogenase enzyme complex.     

Two regions of an avian hepadnavirus RNA pregenome are required in cis for encapsidation.

We have constructed a series of deletion mutants spanning the genome of duck hepatitis B virus in order to determine which regions of the viral genome are required in cis for packaging of the pregenome into capsid particles. Deletion of sequences within either of two nonadjacent regions prevented replication of the mutant viral genomes expressed in a permissive avian hepatoma cell line in the presence of functionally active viral core and P proteins. Extraction of RNA from cells transfected with these replication-defective mutants showed that the mutants retained the capacity to be transcribed into a pregenomic-size viral RNA, but that these RNA species were not packaged into viral capsids. The two regions defined by these deletions are located 36 to 126 (region I) and 1046 to 1214 (region II) nucleotides downstream of the 5' end of the pregenome and contain sequences which are required in cis for encapsidation of the duck hepatitis B virus pregenome.     

The a2m(r-Pa-Pu) Allele of the En-Controlling Element System in Maize

The Enhancer (En) controlling element system in maize includes numerous alleles, one of which, a2m(r-pa-pu), was recovered as a derivative of the a2m(1 1511) allele. The behavior and composition of this allele is described. In the presence of the independently inherited regulatory element, En, the a2m(r-pa-pu) allele mutates and expresses in the aleurone purple, pale and colorless sectors within a colorless background. In the absence of En, the pigmentation is uniformly pale. Colored and colorless germinal mutations of the a2m(r-pa-pu) allele were recovered, although the derivative alleles tested did not respond to En. Uniformly colored derivatives, designated A2', arise only from states of a2m(r-pa-pu) that respond to an active En at relatively early stages in plant development, suggesting that somatic and germinal mutations may arise through similar events. Mutations to A2-expressing alleles appear to occur relatively late in plant development, that is, at or near meiosis, since recognizable mutant sectors do not appear on testcross ears. Kernels exhibiting such mutants are randomly distributed over the ear. The resulting A2' alleles express pigmentation ranging in a continual sequential series from dark pale to intense purple.     

Detection of protein similarities using nucleotide sequence databases.

A simple procedure is described for finding similarities between proteins using nucleotide sequence databases. The approach is illustrated by several examples of previously unknown correspondences with important biological implications: Drosophila elongation factor Tu is shown to be encoded by two genes that are differently expressed during development; a cluster of three Drosophila genes likely encode maltases; a flesh-fly fat body protein resembles the hypothesized Drosophila alcohol dehydrogenase ancestral protein; an unknown protein encoded at the multifunctional E. coli hisT locus resembles aspartate beta-semialdehyde dehydrogenase; and the E. coli tyrR protein is related to nitrogen regulatory proteins. These and other matches were discovered using a personal computer of the type available in most laboratories collecting DNA sequence data. As relatively few sequences were sampled to find these matches, it is likely that much of the existing data has not been adequately examined.     

Attenuation of Sindbis virus neurovirulence by using defined mutations in nontranslated regions of the genome RNA.

We examined a panel of Sindbis virus mutants containing defined mutations in the 5' nontranslated region of the genome RNA, in the 3' nontranslated region, or in both for their growth in cultured cells and virulence in newborn mice. In cultured cells, these viruses all had defects in RNA synthesis and displayed a wide range of growth rates. The growth properties of the mutants were often very different in mouse cells from those in chicken cells or in mosquito cells. We hypothesize that host factors, presumably proteins, interact with these nontranslated regions to promote viral replication and that the mammalian protein and the chicken or mosquito protein are sufficiently divergent that alterations in the viral RNA sequence can affect the interactions with these different host proteins in different ways. Some of the mutants were temperature sensitive for plaque formation, whereas one mutant was slightly cold sensitive in its growth in chicken cells. Upon inoculation into mice, viruses that grew well in cultured mouse cells retained their virulence, but mice that succumbed usually had extended survival times. One virulent mutant that grew slightly less well in cultured mouse cells than did the parental virus produced eight times as much virus in mouse brain following intracerebral inoculation, suggesting that changes in these regulatory regions may have tissue-specific as well as host-specific effects. Viruses that were severely crippled in their growth in mouse cells in culture were usually, but not always, attenuated in their virulence. In particular, temperature sensitivity was correlated with attenuation. The effect of two mutations was found to be cumulative, and double mutants that contained mutations in both the 5' and 3' nontranslated regions were more attenuated than was either single mutant. Three of four double mutants tested were severely crippled for virus production in cultured cells and were avirulent for mice, even when inoculated intracerebrally.     

Two related Epstein-Barr virus membrane proteins are encoded by separate genes.

The structures of the 2.3- and 2.0-kilobase Epstein-Barr virus (EBV) mRNAs, partially encoded within the EcoRI J fragment DNA of the viral genome, were determined by analysis of their cDNAs. Both mRNAs are transcribed across the fused terminal repeats of the EBV episome and consist of nine exons. The mRNAs are transcribed from different promoters and have a unique 5' exon from the U5 region of the genome but eight common exons from the U1 region. One principal open reading frame is present in each mRNA and is predicted to encode 54,000- and 40,000-dalton integral membrane proteins. This result was confirmed by in vitro translation of RNAs in the presence of canine pancreatic microsomes. The 2.3-kilobase mRNA is not expressed in Raji cells, owing to the deletion of the 5' regulatory and coding region of this gene, whereas neither mRNA is expressed in Namalwa cells, owing to inactivation as a result of integration of the EBV genome via the terminal repeats. Since these mRNAs are readily detected in largely latently infected cells and do not increase in abundance with EBV replication, these putative latent-infection membrane proteins are tentatively designated LMP-2A and LMP-2B, respectively.     

The OPI1 gene of Saccharomyces cerevisiae, a negative regulator of phospholipid biosynthesis, encodes a protein containing polyglutamine tracts and a leucine zipper

In Saccharomyces cerevisiae, recessive mutations at the OPI1 locus result in constitutively derepressed expression of inositol 1-phosphate synthase, the product of the INO1 gene. Many of the other enzymes involved in phospholipid biosynthesis are also expressed at high derepressed levels in opi1 mutants. Thus, the OPI1 gene is believed to encode a negative regulator that is required to repress a whole subset of structural genes encoding for phospholipid biosynthetic enzymes. In this study, the OPI1 gene was mapped to chromosome VIII and cloned. When transformed into an opi1 mutant, the cloned DNA was capable of complementing the mutant phenotype and restoring correct regulation to the INO1 structural gene. Construction of two opi1 disruption alleles and subsequent genetic analysis of strains bearing these alleles confirmed that the cloned DNA was homologous to the genomic OPI1 locus. Furthermore, the OPI1 gene was found to be nonessential to the organism since mutants bearing the null allele were viable and exhibited a phenotype similar to that of previously isolated opi1 mutants. Similar to other opi1 mutants, the opi1 disruption mutants accumulated INO1 mRNA constitutively to a level 2-3-fold higher than that observed in wild-type cells. The cloned OPI1 gene was sequenced, and translation of the open reading frame predicted a protein composed of 404 amino acid residues with a molecular weight of 40,036. The predicted Opi1 protein contained a well defined heptad repeat of leucine residues that has been observed in other regulatory proteins. In addition, the predicted protein contained polyglutamine residue stretches which have also been reported in yeast genes having regulatory functions. Sequencing of opi1 mutant alleles, isolated after chemical mutagenesis, revealed that several were the result of a chain termination mutation located within the largest polyglutamine residue stretch.     

Identification of domains of the v-crk oncogene product sufficient for association with phosphotyrosine-containing proteins.

The oncogene product of the avian sarcoma virus CT10, P47gag-crk, contains the SH2, SH2', and SH3 domains and binds proteins in a phosphotyrosine (ptyr)-dependent manner. In this study, we have determined the region of P47gag-crk essential for binding to ptyr-containing proteins. Mutant P47gag-crk proteins expressed in Escherichia coli that have the intact SH2 and SH2' regions retained the capacity to bind ptyr-containing proteins obtained from cells transformed by crk and src. The deletion of SH2 resulted in the loss of binding activity. Other mutants that have altered SH2 or SH2' bound few, if any, of the ptyr-containing proteins. Those mutants that bound ptyr-containing proteins associated with tyrosine kinase activity. We also found that polypeptides containing SH2, SH2', and SH3 of p60v-src and p60c-src associated with ptyr-containing proteins from crk-transformed cells. Thus, the SH2 and SH2' domains of P47gag-crk are responsible for their binding to ptyr-containing proteins.     

Two Alleles of Maize ALCOHOL DEHYDROGENASE 1 Have 3' Structural and Poly(a) Addition Polymorphisms

Two standard electrophoretic alleles of the maize alcohol dehydrogenase 1 locus (Adh1-1S and Adh1-1F) have been isolated and characterized. Restriction endonuclease mapping shows that a region of less than 5 kb is conserved in both alleles and is flanked both 5' and 3' by regions highly polymorphic for restriction sites. Nucleotide sequence comparison of these two alleles reveals that polymorphism in the 3' flanking region is due to rearrangements including tandem duplications, a transposable element-like insertion and a deletion. S1 nuclease analysis shows that both the Adh1-1S and the Adh1-1F alleles contain multiple poly(A) addition sites; four sites are observed for the Adh1-1S alleles and seven sites for the Adh1-1F allele. Only two of these poly(A) addition sites appear to be identical in the two alleles. No consensus signal for poly(A) addition is observed near any of these sites.