Altered SOS induction associated with mutations in recF, recO and recR

The SOS system of Escherichia coli aids survival following damage to DNA by promoting DNA repair while cell division is delayed. Induction of the SOS response is dependent on RecA and also on the product of recF. We show that normal induction also requires the products of recO and recR. SOS induction was monitored using a sfiA-lacZ fusion strain. Induction was delayed to a similar degree by mutation in recF, recO or recR. A similar effect was observed following overexpression of RecR from a recombinant recR ⁺plasmid. We show that the overexpression of RecR also reduces the UV resistance of a recBC sbcBC strain and of a sfiA strain, but not of a rec ⁺ sfiA ⁺strain. The implications of these data for the kinetics of DNA repair are discussed.     

Recombinant cyclin E expression activates proliferation and obviates surface attachment of chinese hamster ovary (CHO) cells in protein-free medium

Exogenous growth factors normally required in cell culture activate cell proliferation via the molecular controls of cell-cycle progression. Highly differing influences of mitogenic stimulation of Chinese hamster ovary (CHO) cells by insulin and basic fibroblast growth factor(bFGF) have been clearly observed in a defined protein-free medium. CHO K1 cells stimulated only with insulin grow with flattened cell morphology and extensive cell-cell contact, whereas stimulation with only bFGF or bFGF plus insulin results in loss of cell-cell contact and a transformed and rounded-up morphology. Compared with insulin-stimulated cells, bFGF-stimulated cells exhibit a relatively long G1, and short S phase, and contain higher levels of cyclin E. Observation of elevated levels of cyclin E in wild-type CHO K1 cells mitogenically stimulated by basic fibroblast growth factor motivated transfection of these cells by a cyclin E expression vector. These transfectants grew rapidly in protein-free basal medium and had similar cyclin b levels, distributions of nuclear cell-cycle times, and cell morphologies as bFGF-stimutated CHO K1 culture. Metabolic engineering of cell-cycle regulation thus bypasses exogenous growth factor requirements, addressing a priority objective in economical, reproducible, and safe biopharmaceutical manufacturing. (c) 1995 John Wiley & Sons Inc.     

Improvement of Escherichia coli microaerobic oxygen metabolism by Vitreoscilla hemoglobin: New insights from NAD(P)H fluorescence and culture redox potential

On-line NAD(P)H fluorescence and culture redox potential (CRP) measurements were utilized to investigate the role of Vitreoscilla hemoglobin (VHb) in perturbing oxygen metabolism of microaerobic Escherichia coli Batch cultures of a VHb-synthesizing E. coli strain and the iso-genic control under fully aerated conditions were subject to several high/low oxygen transitions, and the NAD(P)H fluorescence and CRP were monitored during these passages. The presence of VHb decreased the rate of net NAD(P)H generation by 2.4-fold under diminishing oxygen tension. In the absence of aeration, the strain producing VHb maintained a steady NAD(P)H level 1.8-fold less than that of the control, indicating that the presence of VHb keeps E. coli in a more oxidized state under oxygen-limited conditions. Estimated from CRP, the oxygen uptake rates near anoxia were 25% higher for cells with VHb than those without. These results suggest that VHb-expressing cells have a higher microaerobic electron transport chain turnover rate. To examine how NAD(P)H utilization of VHb-expressing cells responds to rapidly changing oxygen tension, which is common in large-scale fermentations, we pulsed air intermittently into a cell suspension and recorded the fluorescence response to the imposed dissolved oxygen (DO) fluctuation. Relative to the control, cells containing VHb had a sluggish fluorescence response to sudden changes of oxygen tension, suggesting that VHb buffers intracellular redox perturbations caused by extracellular DO fluctuations.(c) John Wiley & Sons, Inc.     

Gene inactivation in the plant pathogen Glomerella cingulata: three strategies for the disruption of the pectin lyase gene pnIA

The feasibility of performing routine transformation-mediated mutagenesis in Glomerella cingulata was analysed by adopting three one-step gene disruption strategies targeted at the pectin lyase gene pnIA. The efficiencies of disruption following transformation with gene replacement- or gene truncation-disruption vectors were compared. To effect replacement-disruption, G. cingulata was transformed with a vector carrying DNA from the pnlA locus in which the majority of the coding sequence had been replaced by the gene for hygromycin B resistance. Two of the five transformants investigated contained an inactivated pnlA gene (pnlA ^– );both also contained ectopically integrated vector sequences. The efficacy of gene disruption by transformation with two gene truncation-disruption vectors was also assessed. Both vectors carried a 5and 3truncated copy of the pnlA coding sequence, adjacent to the gene for hygromycin B resistance. The promoter sequences controlling the selectable marker differed in the two vectors. In one vector the homologous G. cingulata gpdA promoter controlled hygromycin B phosphotransferase expression (homologous truncation vector), whereas in the second vector promoter elements were from the Aspergillus nidulans gpdA gene (heterologous truncation vector). Following transformation with the homologous truncation vector, nine transformants were analysed by Southern hybridisation; no transformants contained a disrupted pnlA gene. Of nineteen heterologous truncation vector transformants, three contained a disrupted pnlA gene; Southern analysis revealed single integrations of vector sequence at pnlA in two of these transformants. pnlA mRNA was not detected by Northern hybridisation in pnlA-transformants. pnlA-transformants failed to produce a PNLA protein with a pI identical to one normally detected in wild-type isolates by silver and activity staining of isoelectric focussing gels. Pathogenesis on Capsicum and apple was unaffected by disruption of the pnlA gene, indicating that the corresponding gene product, PNLA, is not essential for pathogenicity. Gene disruption is a feasible method for selectively mutating defined loci in G. cingulata for functional analysis of the corresponding gene products.     

Genetical and physical assignments of equine microsatellites—first integration of anchored markers in horse genome mapping

Twenty equine microsatellites were isolated from a genomic phage library, and their genetical and physical localization was sought by linkage mapping and fluorescent in situ hybridization (FISH). Nineteen of the markers were found to be polymorphic with, in most cases, heterozygosities exceeding 50%. The markers were mapped in a Swedish reference family for gene mapping, comprising eight half-sib families from Standardbred and Icelandic horse sires. Segregation was analyzed against a set of 35 other markers typed in the pedigree. Thirteen of the microsatellites showed linkage to at least one other marker, with a total of 21 markers being involved in these linkages. In parallel, 18 of the microsatellites could be assigned to their chromosomal region by FISH. These assignments involved eight equine autosomes: ECA1, 2, 4, 6, 9, 10, 15, and 16. The genetical and physical mappings revealed by this study represent a significant extension of the current knowledge of the equine genome map. Received: 24 September 1996 / Accepted: 1 December 1996     

Regulation, replication, and integration functions of the Vibrio cholerae CTXφ are encoded by region RS2

CTXφ is a filamentous phage that encodes cholera toxin, one of the principal virulence factors of Vibrio cholerae . CTXφ is unusual among filamentous phages because it can either replicate as a plasmid or integrate into the V. cholerae chromosome at a specific site. The CTXφ genome has two regions, the 'core' and RS2. Integrated CTXφ is frequently flanked by an element known as RS1 which is related to RS2. The nucleotide sequences of RS2 and RS1 were determined. These related elements contain three nearly identical open reading frames (ORFs), which in RS2 were designated rstR , rstA2 and rstB2 . RS1 contains an additional ORF designated rstC . Functional analyses indicate that rstA2 is required for CTXφ replication and rstB2 is required for CTXφ integration. The amino terminus of RstR is similar to the amino termini of other phage-encoded repressors, and RstR represses the expression of rstA2 . Although genes with related functions are clustered in the genome of CTXφ in a way similar to those for other filamentous phages, the CTXφ RS2-encoded gene products mediating replication, integration and repression appear to be novel.     

Population ecology of the white-nosed coati (Nasua narica) on Barro Colorado Island, Panama

The white-nosed coati, Nasua narica , is a common Neotropical carnivore with a social structure of band-living adult females and solitary adult males. A coati population on Barro Colorado Island, Panama, was studied over a four-year period by mark-recapture, radiotelemetry. and direct observation of habituated individuals. The population density was approximately 51.5 individuals/km² and the sex ratio was 1:1. Band size varied from six to 26 individuals (mean = 15.3) with extensive fluctuation within and between years. Mean foraging group size was smaller (7.2 individuals) than population group size, and fluctuated with food availability, synchronous parturition, and the emigration of mature males. Mean home-range size of six bands was 0.33 km², and ranges of adjacent bands overlapped from 0–66%. One band fissioned during the study; however, the resulting bands did not disperse from the original home range. Seven adult males had a mean home-range size of 0.37 km², each extensively overlapping the home ranges of several other males. Observations of 10 adult males whose natal bands were known indicate that when males disperse they do not simultaneously leave the band's home range. Rather, their home ranges remain within or broadly overlapping those of their natal bands. This dispersal pattern is unusual within the order Carnivora.     

Rco-3, a Gene Involved in Glucose Transport and Conidiation in Neurospora Crassa

Macroconidiation in Neurospora crassa is influenced by a number of environmental cues, including the nutritional status of the growing organism. Conidia formation is normally observed when the fungus is exposed to air. However, carbon limitation can induce conidiation in mycelia submerged in an aerated liquid medium. A mutant was previously isolated that could conidiate in submerged culture without imposing nutrient limitation and the gene responsible for this phenotype (rco-3) has now been cloned. RCO3 exhibits sequence similarity to members of the sugar transporter gene superfamily, with greatest similarity to glucose transporters of yeast. Consistent with this structural similarity, we find that glucose transport activity is altered in the mutant. However, growth of the mutant in media containing alternate carbon sources does not suppress conidiation in submerged culture. The properties of the mutant suggest that RCO3 is required for expression of glucose transport activity, glucose regulation of gene expression, and general carbon repression of development.     

Targeting Determinants and Proposed Evolutionary Basis for the Sec and the Delta pH Protein Transport Systems in Chloroplast Thylakoid Membranes

Transport of proteins to the thylakoid lumen is accomplished by two precursor-specific pathways, the Sec and the unique Delta pH transport systems. Pathway selection is specified by transient lumen-targeting domains (LTDs) on precursor proteins. Here, chimeric and mutant LTDs were used to identify elements responsible for targeting specificity. The results showed that: (a) minimal signal peptide motifs consisting of charged N, hydrophobic H, and cleavage C domains were both necessary and sufficient for pathway-specific targeting; (b) exclusive targeting to the Delta pH pathway requires a twin arginine in the N domain and an H domain that is incompatible with the Sec pathway; (c) exclusive targeting to the Sec pathway is achieved by an N domain that lacks the twin arginine, although the twin arginine was completely compatible with the Sec system. A dual-targeting signal peptide, constructed by combining Delta pH and Sec domains, was used to simultaneously compare the transport capability of both pathways when confronted with different passenger proteins. Whereas Sec passengers were efficiently transported by both pathways, Delta pH passengers were arrested in translocation on the Sec pathway. This finding suggests that the Delta pH mechanism evolved to accommodate transport of proteins incompatible with the thylakoid Sec machinery.     

The Evolution of MHC Diversity by Segmental Duplication and Transposition of Retroelements

Sequence analysis of a 237 kb genomic fragment from the central region of the MHC has revealed that the HLA-B and HLA-C genes are contained within duplicated segments peri-B (53 kb) and peri-C (48 kb), respectively, and separated by an intervening sequence (IF) of 30 kb. The peri-B and peri-C segments share at least 90% sequence homology except when interrupted by insertions/deletions including Alu, L1, an endogenous retrovirus, and pseudogenes. The sequences of peri-B, IF, and peri-C were searched for the presence of Alu elements to use as markers of evolution, chromosomal rearrangements, and polymorphism. Of 29 Alu elements, 14 were identified in peri-B, 11 in peri-C, and 4 in IF. The Alu elements in peri-B and peri-C clustered phylogenetically into two clades which were classified as ``preduplication' and ``postduplication' clades. Four Alu J elements that are shared by peri-B and peri-C and are flanked by homologous sequences in their paralogous locations, respectively, clustered into a ``preduplication' clade. By contrast, the majority of Alu elements, which are unique to either peri-B or peri-C, clustered into a postduplication clade together with the Alu consensus subfamily members ranging from platyrrhine-specific (Spqxcg) to catarrhine-specific Alu sequences (Y). The insertion of platyrrhine-specific Alu elements in postduplication locations of peri-B and peri-C implies that these two segments are the products of a duplication which occurred in primates prior to the divergence of the New World primate from the human lineage (35–44 mya). Examination of the paralogous Alu integration sites revealed that 9 of 14 postduplication Alu sequences have produced microsatellites of different length and sequence within the Alu 3′-poly A tail. The present analysis supports the hypothesis that HLA-B and HLA-C genes are products of an extended segmental duplication between 44 and 81 million years ago (mya), and that subsequent diversification of both genomic segments occurred because of the mobility and mutation of retroelements such as Alu repeats. Received: 21 May 1997 / Accepted: 9 July 1997