Characterization of recognition sites on bacteriophage HP1c1 DNA which interact with the DNA uptake system of Haemophilus influenzae Rd

The 32.4-kb genome of the Haemophilus influenzae bacteriophage HP1c1 contains at least twelve sites, each conferring high affinity for the DNA uptake system of transformable H. influenzae Rd. Five of these high-affinity sites have been located and their nucleotide sequences determined. Three sites contained a contiguous 9-bp sequence identical to the first nine residues of the 11-bp site previously identified as conferring high affinity for the H. influenzae transformation receptor to DNA fragments. The remaining two sites contained complete 11-bp sequences. In contrast, an HP1c1 restriction fragment containing a sequence identical to the final nine residues of the 11-bp uptake site exhibits only a low affinity for the DNA uptake system. An 8-bp sequence consisting of the first eight residues of the 11-bp site was 1% as active as the longer, high-affinity sites. Thus the first 9-bp of the 11-bp site are sufficient to direct high-affinity uptake, while the first 8-bp or the distal 9-bp are not. These results provide an initial assessment of the relative contributions of the individual residues constituting the 11-bp site to the apparent affinity of DNA fragments for the receptor of Haemophilus transformation.     

Two novel gene orders and the role of light-strand replication in rearrangement of the vertebrate mitochondrial genome

Two novel mitochondrial gene arrangements are identified in an agamid lizard and a ranid frog. Statistical tests incorporating phylogeny indicate a link between novel vertebrate mitochondrial gene orders and movement of the origin of light-strand replication. A mechanism involving errors in light-strand replication and tandem duplication of genes is proposed for rearrangement of vertebrate mitochondrial genes. A second mechanism involving small direct repeats also is identified. These mechanisms implicate gene order as a reliable phylogenetic character. Shifts in gene order define major lineages without evidence of parallelism or reversal. The loss of the origin of light-strand replication from its typical vertebrate position evolves in parallel and, therefore, is a less reliable phylogenetic character. Gene junctions also evolve in parallel. Sequencing across multigenic regions, in particular transfer RNA genes, should be a major focus of future systematic studies to locate novel gene orders and to provide a better understanding of the evolution of the vertebrate mitochondrial genome.      

Cloning and expression of draTG genes from Azospirillum lipoferum

A genomic library of Azospirillum lipoferum was constructed with phage lambda EMBL4 as vector. From this library, the genes encoding dinitrogenase reductase ADP-ribosyltransferase (DRAT), draT, and dinitrogenase reductase-activating glycohydrolase (DRAG), draG, were cloned by hybridization with the heterologous probes of Rhodospirillum rubrum. As in R. rubrum, draT is located between draG and nifH, the gene encoding dinitrogenase reductase (a substrate for the DRAG/DRAT system). In the crude extract of Escherichia coli harboring the expression vector for this region, DRAT and DRAG enzyme activities were detected, confirming the identity of the cloned genes. Southern hybridization with genomic DNA from different Azospirillum spp., demonstrated a correlation between observable draTG hybridization and the biochemical demonstration of this covalent modification system.     

Glutamate catabolism in Rhizobium meliloti

The pathway by which glutamate is degraded as a carbon source has not previously been elucidated, but enzymatic analysis of Rhizobium meliloti CMF1 indicated that both glutamate dehydrogenase (GDH) and gamma-aminobutyrate (GABA) bypass activities were present in free living cells. However, when similar studies were performed on R. meliloti CMF1 bacteroids, isolated from alfalfa nodules, only GABA bypass activities were detectable. Both GDH and GABA bypass activities were influenced by the carbon source provided, with maximum activities being detected when glutamate was present as sole carbon and nitrogen source. Addition of a second carbon source, such as succinate, to the growth medium did not influence GDH activity but substantially decreased levels of the first enzyme of the GABA bypass, glutamate decarboxylase (GDC). Cyclic adenosine 3′5′-monophosphate (cAMP) failed to increase GDC activities in R. meliloti CMF1 cells grown in the presence of an additional carbon source. It is proposed that the GABA bypass is a major mechanism of glutamate carbon degradation in R. meliloti CMF1, a system whose enzymatic activities are influenced by the nature of the carbon source present in the growth environment.     

Transposon tagging of the sulfur gene of tobacco using engineered maize Ac/Ds elements

The Sulfur gene of tobacco is nuclearly encoded. A Su allele at this locus acts as a dominant semilethal mutation and causes reduced accumulation of chlorophyll, resulting in a yellow color in the plant. An engineered transposon tagging system, based upon the maize element Ac/Ds, was used to mutate the gene. High frequency of transposon excision from the Su locus produced variegated sectors. Plants regenerated from the variegated sector exhibited a similar variegated phenotype. Genetic analyses showed that the variegation was always associated with the transposase construct and the transposon was linked to the Su locus. Sequences surrounding the transposon were isolated, and five revertant sectors possessed typical direct repeats following Ds excisions. These genetic and molecular data are consistent with the tagging of the Su allele by the transposon.     

Evolutionary relationships among the male and female mitochondrial DNA lineages in the Mytilus edulis species complex

A novel form of mitochondrial DNA (mtDNA) inheritance has previously been documented for the blue mussel (Mytilus edulis). Female mussels inherit their mtDNA solely from their mother while males inherit mtDNA from both their mother and their father. In males, the paternal mtDNA is preferentially amplified so that the male gonad is highly enriched for the paternal mtDNA that is then transmitted from fathers to sons. We demonstrate that this mode of mtDNA inheritance also operates in the closely related species M. galloprovincialis and M. trossulus. The evolutionary relationship between the male and female mtDNA lineages is estimated by phylogenetic analysis of 455 nucleotides from the large subunit ribosomal RNA gene. We have found that the male and female lineages are highly divergent; the divergence of these lineages began prior to the speciation of the three species of blue mussels. Further, the separation between the male and female lineages is estimated to have occurred between 5.3 and 5.7 MYA.