Physical mapping of the att-N region of coliphage lambda: Apparent oversaturation of coding capacity in the gam-ral segment

Employing a series of λbio deletion mutants, the genes int, xis, Ea22, exo, bet, gam, kil, cIII, Ea10, ral and N and their known protein products have been ordered in respect to the physical map of λ, as constructed by electron microscopy of heteroduplexes between the λbio's and λattR-attL's (λatt²imm21 or λatt²imm434). Throughout most of this region the coding capacity is about saturated, as based on the known sizes of the λ proteins and the positions of the genetic and physical endpoints of the bio deletions. One baffling exception is the gam-kil-cIII segment, which cannot accommodate all three of the corresponding proteins. The second exception is the region between genes ral and N, which could code for a protein about 25,000 daltons, but for which no proteins have been identified. The space available for gene N suggests that its product must be a protein of no more than 15,000 daltons in size. Physical mapping of the bio386 deletion has established that proteins Ea42(int), Ea32(xis ?) and Ea22 are encoded in that order.     

pHG276: A multiple cloning site pBR322 copy number vector expressing a functional lacα peptide from the bacteriophage lambda PR promoter

The bacteriophage lambda early promoter P_R has been used to direct the synthesis of lacα in a plasmid containing the multiple cloning site of pUC8. The copy number of the plasmid is controlled by the rop(rom) gene and the plasmid incorporates the cI⁸⁵⁷ gene for temperature regulation of lacα expression. Isolation of recombinant derivatives with DNA inserts in the multiple cloning region can be identified by insertional inactivation of lacα and consequently, a Lac⁻ phenotype in a host carrying the M15 deletion of lac. The potential of pHG276 as a fully regulated expression vector is examined.     

Total gene deletions and mutant frequency of the HPRT gene as indicators of radiation exposure in Chernobyl liquidators

This study was conducted to determine the utility of deletion spectrum and mutant frequency (MF) of the hypoxanthine phosphoribosyl transferase gene (HPRT) as indicators of radiation exposure in Russian Liquidators who served in 1986 or 1987 in the clean up effort following the nuclear power plant accident at Chernobyl. HPRT MF was determined using the cloning assay for 117 Russian Controls and 122 Liquidators whose blood samples were obtained between 1991 and 1998. Only subjects from whom mutants were obtained for deletion analysis are included. Multiplex PCR analysis was performed on cell extracts of 1080 thioguanine resistant clones from Controls and 944 clones from Liquidators. Although the deletion spectra of Liquidators and Controls were similar overall, the Liquidator deletion spectrum was heterogeneous over time. Most notable, the proportion of total gene deletions was higher in 1991–1992 Liquidators than in Russian Controls (χ²=10.5, p=0.001) and in 1993–1994 Liquidators (χ²=8.3, p=0.004), and was marginally elevated relative to 1995–1996 Liquidators (χ²=3.3, p=0.07). This type of mutation has been highly associated with radiation exposure. Total gene deletions were not increased after 1992. Band shift mutations were also increased in the 1991–1992 Liquidators but were associated with increased MF of both Liquidators and Controls (p=0.009), not with increased MF in 1991–1992 Liquidators (p=0.7), and hence are not believed to be associated with radiation exposure. Regression analysis demonstrated that relative to Russian Controls HPRT MF was elevated in Liquidators overall when adjusted for age and smoking status (37%, p=0.0001), and also was elevated in Liquidators sampled in 1991–1992 (72%, p=0.0076), 1993–1994 (22%, p=0.037), and 1995–1996 (62%, p=0.0001). In summary, HPRT MF was found to be the more sensitive and persistent indicator of radiation exposure, but the specificity of total gene deletions led to detection of probable heterogeneity of radiation exposure within the exposed population.     

Recombination between repeats in Escherichia coli by a recA-independent, proximity-sensitive mechanism

We have examined the influence of proximity on the efficiency of recombination between repeated DNA sequences in Escherichia coli. Our experiments have employed a plasmid-based assay to detect deletions between direct repeats of 100 bp. The rate of deletion of the juxtaposed direct repeats was reasonably high at 6 × 10^–5 per cell. A comparison of recA+ and recA mutant strains showed that these deletion events are primarily the result of recA-independent recombination at these homologous sequences. Random restriction fragments of yeast or E. coli genomic DNA were used to separate the two repeats. Deletion rates decreased over two orders of magnitude with increasing separation of up to 7 kb. There was a surprisingly strong effect of even short sequence separations, with insertions of a few hundred base pairs exhibiting 10-fold reductions of deletion rates. No effect of recA on the efficiency of deletion was observed at any distance between repeats.     

Further characterization of the respiratory deficient dum-1 mutation of Chlamydomonas reinhardtii and its use as a recipient for mitochondrial transformation

Summary The respiratory deficient dum-1 mutant of Chlamydomonas reinhardtii fails to grow in the dark because of a terminal 1.5 kb deletion in the linear 15.8 kb mitochondrial genome, which affects the apocytochrome b (CYB) gene. In contrast to the wild type where only mitochondrial genomes of monomer length are observed, the dum-1 genomes are present as a mixture of monomer and dimer length molecules. The mutant dimers appear to result from head-to-head fusions of two deleted molecules. Furthermore, mitochondrial genomes of dum-1 were also found to be unstable, with the extent of the deletion varying among single cell clones from the original mutant population. The dum-1 mutant also segregates, at a frequency of ca. 4% per generation, lethal minute colonies in which the original deletion now extends at least into the adjacent gene encoding subunit four of NAD dehydrogenase (ND4). We have used the dum-1 mutant as a recipient to demonstrate stable mitochondrial transformation in C. reinhardtii employing the biolistic method. After 4 to 8 weeks dark incubation, a total of 22 respiratory competent colonies were isolated from plates of dum-1 cells bombarded with C. reinhardtii mitochondrial DNA (frequency 7.3 × 10^–7) and a single colony was isolated from plates bombarded with C. smithii mitochondrial DNA (frequency 0.8 × 10^–7). No colonies were seen on control plates (frequency < 0.96 × 10^–9). All transformants grew normally in the dark on acetate media; 22 transformants were homoplasmic for the wild-type mitochondrial genome typical of the C. reinhardtii donor. The single transformant obtained from the C. smithii donor had a recombinant mitochondrial genome containing the donor CYB gene and the diagnostic HpaI and XbaI restriction sites in the gene encoding subunit I of cytochrome oxidase (COI) from the C. reinhardtii recipient. The characteristic deletion fragments of the dum-1 recipient were not detected in any of the transformants.     

Functional expression of N-terminal truncated α-subunits of Na,K-ATPase in Xenopus laevis oocytes

N-terminal deletion mutants of Na,K-ATPase α₁ isoforms initiating translation at Met³⁴ (α₁T₁) or at Met⁴³ (α₁T₂) were expressed in X. laevis oocytes. Compared to β₃ cRNA injected controls, the co-expression of α₁wt, α₁T₁, α₁T₂ with β₃ subunits results in a 2- to 3-fold increase of ouabain binding sites, parallelled by a concomitant increase in Na,K-pump current. The apparent K for potassium activation of the α₁T₂/β₃ Na,K-pumps is significantly higher than that of the α₁wt/β₃ or α₁T₁/β₃ Na,K-pumps expressed at the cell surface. Total deletion of the lysine-rich N-terminal domain thus allows the expression of active Na,K-pump but with distinct cation transport properties.     

The GEF1 gene of Saccharomyces cerevisiae encodes an integral membrane protein; mutations in which have effects on respiration and iron-limited growth

We have isolated a new class of respiration-defective, i.e petite, mutants of the yeast Saccharomyces cerevisiae. Mutations in the GEF1 gene cause cells to grow slowly on rich media containing carbon sources utilized by respiration. This phenotype is suppressed by adding high concentrations of iron to the growth medium. Gef1 ^– mutants also fail to grow on a fermentable carbon source, glucose, when iron is reduced to low concentrations in the medium, suggesting that the GEF1 gene is required for efficient metabolism of iron during growth on fermentable as well as respired carbon sources. However, activity of the iron uptake system appears to be unaffected in gef1 ^– mutants. Fe(II) transporter activity and regulation is normal in gef1 ^– mutants. Fe(III) reductase induction during iron-limited growth is disrupted, but this appears to be a secondary effect of growth rate alterations. The wild-type GEF1 gene was cloned and sequenced; it encodes a protein of 779 amino acids, 13 possible transmembrane domains, and significant similarity to chloride channel proteins from fish and mammals, suggesting that GEF1 encodes an integral membrane protein. A gef1 ^– deletion mutation generated in vitro and introduced into wild-type haploid strains by gene transplacement was not lethal. Oxygen consumption by intact gef1 ^– cells and by mitochondrial fractions isolated from gef1 ^– mutants was reduced 25–50% relative to wild type, indicating that mitochondrial function is defective in these mutants. We suggest that GEF1 encodes a transport protein that is involved in intracellular iron metabolism.     

An Escherichia coli plasmid-based, mutational system in which supF mutants are selectable: Insertion elements dominate the spontaneous spectra

A new system is described to determine the mutational spectra of mutagens and carcinogens in Escherichia coli; data on a limited number (142) of spontaneous mutants is presented. The mutational assay employs a method to select (rather than screen) for mutations in a supF target gene carried on a plasmid. The E. coli host cells (ES87) are lacI⁻ (am26), and carry the lacZΔM15 marker for α-complementation in β-galactosidase. When these cells also carry a plasmid, such as pUB3, which contains a wild-type copy of supF and lacZ-α, the lactose operon is repressed (off). Furthermore, supF suppression of lacl^um26 results in a lactose repressor that has an uninducible, lacl^S genotype, which makes the cells unable to grow on lactose minimal plates. In contrast, spontaneous or mutagen-induced supF⁻ mutations in pUB3 prevent suppresion of lacl^am26 and result in constitutive expression of the lactose operon, which permits growth on lactose minimal plates. The spontaneous mutation frequency in the supF gene is 0.7 and 1.0 × 10⁻⁶ without and with SOS induction, respectively. Spontaneous mutations are dominated by large insertions (67% in SOS-uninduced and 56% in SOS-induced cells), and their frequency of appearance is largely unaffected by SOS induction. These are identified by DNA sequencing to be Insertion Element: IS1 dominates, but IS4, IS5, gamma-delta and IS10 are also obtained. Large deletions also contribute significantly (19% and 15% for - SOS and +SOS, respectively), where a specific deletion between a 10 base pair direct repeat dominates; the frequency of appearance of these mutations also appears to be unaffected by SOS induction. In contrast, SOS induction increases base pairing mutations (13% and 27% for -SOS and +SOS, respectively), The ES87/pUB3 system has many advantages for determining mutational spectra, including the fact that mutant isolation is fast and simple, and the determination of mutational changes is rapid because of the small size of supF.     

The DNA gyrase of Escherichia coli participates in the formation of a spontaneous deletion by recA-independent recombination in vivo

Summary A system for detecting a spontaneous deletion in Escherichia coli was developed and the role of DNA gyrase in deletion formation was studied. A derivative of plac5, AM36, was isolated in which whole pBR322 DNA was inserted in the lacZ gene and 227 by of the lac gene duplicated at both sides of the pBR322 DNA. E. coli lac ^–strains lysogenized by AM36 had a Lac^– phenotype and segregated Lac^– revertants. Sequence analyses showed that the revertant was formed by a deletion that eliminated the inserted pBR322 DNA and one copy of the duplicated segments. The frequency of lac ^– revertant formation was independent of recA function, was increased by oxolinic acid, an inhibitor of DNA gyrase, but was not increased in a lysogen of a nalidixic acid-resistant derivative. The reversion frequencies of temperature sensitive mutants of gyrA gene are 10 to 100 times lower than that of the wild-type strain. These results indicate that the DNA gyrase of E. coli participated in the in vivo deletion formation resulting from the direct repeats.     

Properties of strains of Escherichia coli K12 carrying mutant lex and rec alleles

Summary Strains of Escherichia coli K12 have been constructed which carry the lex-3 mutation in combination with recA56, recB21, or recC22. The lex ^– recA ^–strain is equally as sensitive to ultraviolet light (UV) and ionizing radiation and as recombination-deficient as the corresponding lex ⁺ recA ^–strain, whereas the lex ^– recB ^–and lex ^– recC ^–strains are somewhat more sensitive to UV and ionizing radiation than the corresponding lex ^– rec ⁺strain and have approximately the same recombination deficiencies as the respective lex ⁺ recB ^–and Lex ⁺ recC ^–strains. When cultures of the lex ^– recB ^–and lex ^–recC^–strains are UV-irradiated, they degrade their DNA at the low rate characteristic of lex ⁺ recB ^–and lex ⁺ recC ^–single mutants, in contrast to the high rate of degradation seen with lex ^– rec ⁺single mutants.These results imply that the lex ⁺and recA ⁺products act in the same pathway of DNA repair and that both are needed to limit the DNA breakdown due to the recB ⁺/C⁺nuclease.     

Functional analysis of the plasmid pM4 replicon from Lactobacillus plantarum M4: Determination of the minimal replicon and functionality identification of the putative sso

In order to determine the minimal replicon and the single strand origin (sso) of the plasmid pM4, different fragments of pM4 were amplified by polymerase chain reaction (PCR) and cloned into pBEm, a replication probe vector for Lactobacillus. The deletion analysis results showed that the minimal replicon of pM4 could be determined within a 1280 bp fragment consisting of double strand origin (dso) and rep gene encoding replication protein. Based on plasmid segregation stability assay and its ability to convert single-stranded DNA (ssDNA) to double-stranded DNA (dsDNA) by Southern hybridization, an sso of replication was located at nucleotides −118–92 in the plasmid pM4, about 300 bp upstream of dso. In addition, the host range assay indicated that plasmid pM4 could replicate in L. casei 05–21, L. rhamnosus AS 1.2466^T and L. plantarum 05–19 of all the tested Lactobacillus strains. Analysis of the pM4 replicon will allow its use in constructing a food-grade vector for application in food industry.     

Physical Localization of the Mesoderm Development (mesd) Functional Region

The mesoderm development (mesd) functional interval is essential for primitive streak formation and mesoderm induction. Mesd is defined by overlapping albino (c) deletions on chromosome 7. We have constructed a bacterial artificial chromosome (BAC) contig that spans the mesd functional region. BAC end-sequence identifies three segments that recognize novel expressed sequences. Localization of the proximal breakpoints from Del(7)Tyr^c−3YPSd and Del(7)Tyr^c−112K within the contig defines a deletion interval of 310–350 kb that is essential for mesd function. Importantly, using BAC transgene rescue, we define a 75-kb mesd critical region containing at least one expressed sequence.     

Construction in Escherichia coli K12 of derivatives of sex factor F143 carrying lexB and recA mutations

Summary Various F sex factors have been derived from F143, an episome extending from lysA to pheA. F143 derivatives carrying recA and lexB alleles and also mutations in genes thyA, argA, cysC were constructed as follows. Recombination was used as a means to generate genetically labelled F-primes. Using trimethoprim as agent of counterselection of Thy⁺ cells in thyA ^–/F-thyA ⁺bacteria, it was possible to collect, after transfer, F-primes modified by deletion of the thyA region or recombination between chromosome and episome. F-primes which had spontaneously recombined with the chromosome and integrated chromosomal markers, were also selected by transfer to proper F^– recipients. P1 transduction of a dominant marker allele into a strain homodiploid for a recessive allele was used to construct F-primes carrying mutations introduced by cotransduction. These F-primes have been useful to establish the dominance and complementation pattern of recA and lexB mutations (Morand, Goze and Devoret, accompanying paper; Glickman, Guijt and Morand, accompanying paper).     

Chloramphenicol resistance in Streptomyces coelicolor A3(2): Possible involvement of a transposable element

Summary The transfer of a Chl element, causing resistance to chloramphenicol in Streptomyces coelicolor A3(2), was studied in NF x SCP1^– superfertile crosses. When the Chl element is on the donor side (NF) its transfer to the recombinant cells was virtually total as if the element acted as a second concomitant transfer origin. When the Chl element was on the recipient side (SCP1^–) it was never displaced by the immigrant chromosome even when the region facing chl ⁺ was selected for. A fraction of the original Chl^– mutants presented a requirement for arginine (ArgB^–). A Chl^– mutant gave rise spontaneously to ArgB^– derivatives at high frequency. The same ArgB^– requirement come out at high frequency among Chl^– derivatives from a cross NFChl^– x SCP1^–Chl⁺ in which neither parent required arginine or produced spontaneously arginineless derivatives. It is suggested that the Chl element is a transposable element (Tn) presumably associated with insertion sequences (IS). The insertional inactivation of the Chl element may be accompanied or followed by a deletion in the adjacent ArgB gene.     

Variation in δC values for the seagrass Thalassia testudinum and its relations to mangrove carbon

Carbon isotope ratios (¹³C/¹²C) were measured for the leaves of the seagrass Thalassia testudinum Banks ex König and carbonates of shells collected at the seagrass beds from seven sites along the coast of southern Florida, U.S.A. The δ¹³C values of seagrass leaves ranged from −7.3 to −16.3‰ among different study sites, with a significantly lower mean value for seagrass leaves from those sites near mangrove forests (−12.8 ± 1.1‰) than those far from mangrove forests (−8.3 ± 0.9‰; P < 0.05). Furthermore, seagrass leaves from a shallow water area had significantly lower δ¹³C values than those found in a deep water area (P < 0.01). There was no significant variation in δ¹³C values between young and mature leaves (P = 0.59) or between the tip and base of a leaf blade (P = 0.46). Carbonates of shells also showed a significantly lower mean δ¹³C value in the mangrove areas (−2.3 ± 0.6‰) than in the non-mangrove areas (0.6 ± 0.3‰; P <0.025). In addition, the δ¹³C values of seagrass leaves were significantly correlated with those of shell carbonates (δ¹³C seagrass leaf = −9.1 + 1.3δ¹³C shell carbonate (R² = 0.83, P < 0.01)). These results indicated that the input of carbon dioxide from the mineralization of mangrove detritus caused the variation in carbon isotope ratios of seagrass leaves among different sites in this study.     

Altered Astrocytic Swelling in the Cortex of α-Syntrophin-Negative GFAP/EGFP Mice

Brain edema accompanying ischemic or traumatic brain injuries, originates from a disruption of ionic/neurotransmitter homeostasis that leads to accumulation of K⁺ and glutamate in the extracellular space. Their increased uptake, predominantly provided by astrocytes, is associated with water influx via aquaporin-4 (AQP4). As the removal of perivascular AQP4 via the deletion of α-syntrophin was shown to delay edema formation and K⁺ clearance, we aimed to elucidate the impact of α-syntrophin knockout on volume changes in individual astrocytes in situ evoked by pathological stimuli using three dimensional confocal morphometry and changes in the extracellular space volume fraction (α) in situ and in vivo in the mouse cortex employing the real-time iontophoretic method. RT-qPCR profiling was used to reveal possible differences in the expression of ion channels/transporters that participate in maintaining ionic/neurotransmitter homeostasis. To visualize individual astrocytes in mice lacking α-syntrophin we crossbred GFAP/EGFP mice, in which the astrocytes are labeled by the enhanced green fluorescent protein under the human glial fibrillary acidic protein promoter, with α-syntrophin knockout mice. Three-dimensional confocal morphometry revealed that α-syntrophin deletion results in significantly smaller astrocyte swelling when induced by severe hypoosmotic stress, oxygen glucose deprivation (OGD) or 50 mM K⁺. As for the mild stimuli, such as mild hypoosmotic or hyperosmotic stress or 10 mM K⁺, α-syntrophin deletion had no effect on astrocyte swelling. Similarly, evaluation of relative α changes showed a significantly smaller decrease in α-syntrophin knockout mice only during severe pathological conditions, but not during mild stimuli. In summary, the deletion of α-syntrophin markedly alters astrocyte swelling during severe hypoosmotic stress, OGD or high K⁺.