The hip locus of Escherichia coli affects the frequency of persistence to the lethal consequences of selective inhibition of either DNA or peptidoglycan synthesis. Regulation of the hip operon, which consists of a regulatory region and two genes, hipB and hipA, was examined with strains containing a hip-lac transcriptional fusion placed in single copy at the aft site. Disruption of the hip locus increased activity from the fusion 16-fold. Repression was restored by supplying HipB in trans. HipB was overexpressed and purified. On the basis of gel filtration and cross-linking studies, HipB is a dimer in solution. Sequence analysis revealed that HipB is a Cro-like DNA-binding protein. The interaction of HipB with the hip regulatory region was examined by gel retardation, DNase I protection, and methylation protection studies. HipB binds with a Kapp (K apparent) of 40 pM to four operator sites with the conserved sequence TATCCN8GGATA (N represents any nucleotide). Binding to the operators is nearly simultaneous and appears to be cooperative. Analysis of the role of HipA in the regulation of the hip operon is complicated by the toxicity of HipA in the absence of HipB. Strains disrupted in hipB but not in hipA could not be recovered. Moreover, hipA-containing plasmids cannot be replicated in strains defective in or lacking hipB. HipA is found exclusively in a tight complex with HipB.
Two independent assays capable of measuring the relative in vivo translational step times across a selected codon pair in a growing polypeptide in the bacterium Escherichia coli have been employed to demonstrate that codon pairs observed in protein coding sequences more frequently than predicted (over-represented codon pairs) are translated slower than pairs observed less frequently than expected (under-represented codon pairs). These results are consistent with the findings that translational step times are influenced by codon context and that these context effects are related to the compatabilities of adjacent tRNA isoacceptor molecules on the surface of a translating ribosome. These results also support our previous suggestion that the frequency of one codon next to another has co-evolved with the structure and abundance of tRNA isoacceptors in order to control the rates of translational step times without imposing additional constraints on amino acid sequences or protein structures.While it is known that translational elongation rates are discontinuous and influenced by codon context, the reasons for variations in translational step times across individual codon pairs are not well understood. The prevailing theory has been that translational rates reflect the correlation between the species-specific usage of a given codon and the abundance of its cognate tRNA (Sorensen et al., 1989). However, more recent experimental results support the idea that translation rates are influenced by the compatabilities of adjacent tRNAs in the A-and P-sites on the surface of translating ribosomes (Smith and Yarus, 1989;Yarus and Curran, 1992). In support of this idea, we previously described an extreme, species-specific, codon pair utilization bias in bacteria, yeast, and mammals (Gutman and Hatfield, 1989;Hatfield and Gutman, 1992). We showed that some codon pairs are used in protein coding sequences much more frequently than expected from the usage of the individual codons of these pairs (over-represented codon pairs), and that some codon pairs are observed much less frequently than expected (under-represented codon pairs). Similar results were obtained by Kolaskar and Reddy (1986) who analyzed codon pair bias in the protein coding sequences of Escherichia coli together with nine coliphages.For E. coli, our codon pair utilization analysis (Gutman and Hatfield, 1989;Hatfield and Gutman, 1992) was performed on a collection of 237 nonredundant protein coding sequences containing 75,403 codon pairs taken from the GENBANK data base (Release 40.0). The usage frequencies of the 61 nonterminating codons were determined and used to calculate the expected values for the random occurrence of each of the 3721 (61 2 ) codon pairs in these sequences. The actual occurrence of each codon pair in the data set was tabulated, and these expected and observed values were used to calculate a 2 value for each codon pair (CHISQ1).1 These values represent the degree of bias of codon pair usage, and we arbitrarily identify 2 values associated with u...
A collection of 4457 Saccharomyces cerevisiae mutants deleted for nonessential genes was screened for mutants with increased or decreased mobilization of the gypsylike retroelement Ty3. Of these, 64 exhibited increased and 66 decreased Ty3 transposition compared with the parental strain. Genes identified in this screen were grouped according to function by using GOnet software developed as part of this study. Gene clusters were related to chromatin and transcript elongation, translation and cytoplasmic RNA processing, vesicular trafficking, nuclear transport, and DNA maintenance. Sixty-six of the mutants were tested for Ty3 proteins and cDNA. Ty3 cDNA and transposition were increased in mutants affected in nuclear pore biogenesis and in a subset of mutants lacking proteins that interact physically or genetically with a replication clamp loader. Our results suggest that nuclear entry is linked mechanistically to Ty3 cDNA synthesis but that host replication factors antagonize Ty3 replication. Some of the factors we identified have been previously shown to affect Ty1 transposition and others to affect retroviral budding. Host factors, such as these, shared by distantly related Ty retroelements and retroviruses are novel candidates for antiviral targets.
A set of shuttle vectors was constructed to facilitate expression of genes for metabolic engineering in Saccharomyces cerevisiae. Selectable markers include the URA3, TRP1, MET15, LEU2-d8, HIS3 and CAN1 genes. Differential expression of genes can be achieved as each marker is available on both CEN/ARS-and 2 µ-containing plasmids. Unique restriction sites downstream of TEF1, PGK1 or HXT7-391 promoters and upstream of the CYC1 terminator allow insertion of open-reading frame cassettes for expression. Furthermore, a fragment appropriate for integration into the genome via homologous recombination can be readily generated in a polymerase chain reaction. Vector marker genes are flanked by loxP recognition sites for the CreA recombinase to allow efficient site-specific marker deletion and recycling. Expression and copy number were characterized for representative high-and low-copy vectors carrying the different marker and promoter sequences. Metabolic engineering typically requires the stable introduction of multiple genes and genomic integration is often preferred. This requires an expanded number of stable expression sites relative to standard gene expression studies. This study demonstrated the practicality of polymerase chain reaction amplification of an expression cassette and genetic marker, and subsequent replacement of endogenous retrotransposons by homologous recombination with flanking sequences. Such reporters were expressed comparably to those inserted at standard integration loci. This expands the number of available characterized integration sites and demonstrates that such sites provide a virtually inexhaustible pool of integration targets for stable expression of multiple genes. Together these vectors and expression loci will facilitate combinatorial gene expression for metabolic engineering.
The retrovirus-like element Ty3 of Saccharomyces cerevisiae integrates at the transcription initiation region of RNA polymerase III. To identify host genes that affect transposition, a collection of insertion mutants was screened using a genetic assay in which insertion of Ty3 activates expression of a tRNA suppressor. Fifty-three loci were identified in this screen. Corresponding knockout mutants were tested for the ability to mobilize a galactose-inducible Ty3, marked with the HIS3 gene. Of 42 mutants tested, 22 had phenotypes similar to those displayed in the original assay. The proteins encoded by the defective genes are involved in chromatin dynamics, transcription, RNA processing, protein modification, cell cycle regulation, nuclear import, and unknown functions. These mutants were induced for Ty3 expression and assayed for Gag3p protein, integrase, cDNA, and Ty3 integration upstream of chromosomal tDNA Val(AAC) genes. Most mutants displayed differences from the wild type in one or more intermediates, although these were typically not as severe as the genetic defect. Because a relatively large number of genes affecting retrotransposition can be identified in yeast and because the majority of these genes have mammalian homologs, this approach provides an avenue for the identification of potential antiviral targets.
A complete understanding of the mode of evolution of molecular markers is important for making inferences about different population genetic parameters, especially because a number of studies have reported patterns of allelic variation at molecular markers that are not in agreement with neutral evolutionary expectations. In the present study, house mice (Mus domesticus) from the fourteenth generation of a selection experiment for increased voluntary wheel-running activity were used to test how selection on a complex behavior affects the distribution of allelic variation by examining patterns of variation at six microsatellite and four allozyme loci. This population had a hierarchical structure that allowed for simultaneous testing of the effects of selection and genetic drift on the distribution of allelic variation by comparing observed patterns of allele frequencies and estimates of genetic divergence at multiple hierarchical levels to expectations under models of neutral evolution. The levels of genetic divergence among replicate lines and between selection groups, estimated from microsatellite data or pooled microsatellite and allozyme data, were not significantly different from expectations under neutral evolution. Furthermore, the pattern of change of allele frequencies between the base population and generation 14 was largely in agreement with expectations under neutral evolution (although the PGM locus exhibited a pattern of change within populations that was difficult to explain under neutral evolution). Overall the results generally provide support for the neutral evolution of molecular markers.
BackgroundThe yeast retrotransposon Ty3 forms stable virus-like particles. Gag3, the major structural protein, is composed of capsid, spacer and nucleocapsid domains. The capsid domain of Gag3 was previously modeled as a structure similar to retrovirus capsid.FindingsTwo-hybrid analysis was used to understand the interactions that contribute to particle assembly. Gag3 interacted with itself as predicted based on its role as the major structural protein. The N-terminal subdomain (NTD) of the capsid was able to interact with itself and with the C-terminal subdomain (CTD) of the capsid, but interacted less well with intact Gag3. Mutations previously shown to block particle assembly disrupted Gag3 interactions more than subdomain interactions.ConclusionsThe findings that the NTD interacts with itself and with the CTD are consistent with previous modeling and a role similar to that of the capsid in retrovirus particle structure. These results are consistent with a model in which the Gag3-Gag3 interactions that initiate assembly differ from the subdomain interactions that potentially underlie particle stability.
Multi-colony stimulating factor (Multi-CSF, interleukin-3, IL-3) and granulocyte-CSF (G-CSF) administered concurrently as an intravenous (IV) injection induce a peripheral neutrophilia that is approximately additive in comparison with the neutrophilia induced by IL-3 and G-CSF individually. The bone marrow (BM) at 12 hours is depleted of mature neutrophils and shows a left-shifted myeloid hyperplasia, consistent with the neutrophil-releasing and myeloproliferative activities of both IL-3 and G-CSF individually. The BM at 24 hours shows a replenished reserve of mature neutrophils and a synergistic left-shifted myeloid hyperplasia as compared with IL-3 and G-CSF alone. Daily IV injections of IL-3 plus G-CSF for 1 week also induce an approximately additive daily peripheral neutrophilia. The BM after a week's administration of IL-3 plus G-CSF shows a generalized myeloid hyperplasia with a synergistic increase in mature neutrophils as compared with IL-3 or G- CSF alone. Daily injection of IL-3 plus G-CSF induced a significant decrease in erythroid, lymphoid, and eosinophilic marrow precursors, possibly owing to a myelophthisic effect of the myeloid hyperplasia and despite the fact that IL-3 alone induced a significant erythroid hyperplasia.
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