Formation of type II F-primes from unstable Hfrs and their recA-independent conversion to other F-prime types

Summary Four E. coli Hfr strains, representing stable (Hfr Cavalli), moderately stable (AB312) and unstable (Ra-1, Ra-2) Hfr states, were used in the isolation of a series of F plasmids. Type II Fs were found to be the most prevalent F plasmid formed from all of the Hfrs, while the percentages of tra Fs increased as the stability of the Hfr increased. Two observations suggested that F formation in unstable Hfrs like Ra-2 may proceed through a type II F precursor. First, the major F products of Ra-2 are tra ⁺ type II Fs and, second, other F types (I, II) and classes (tra ⁺, tra) from Ra-2 appeared to be deletion derivatives of a larger F progenitor. By monitoring the molecular changes that occur when the Ra-2 derived type II F pWS200 is transferred from one recA host to another, we have found that all F types and classes can be generated from pWS200 in a recA-independent manner. F sequences involved in the genetic conversions of pWS200 include the oriT locus and the directly repeated junctions of F and chromosomal DNA. A model for the formation of Fs in unstable Hfrs is postulated in which a tra ⁺ type II F primary excision product is seen to be modified, through recA-independent processes, to other F types and classes. This model differs from the current model of F formation in that independent excision events from the Hfr chromosome are not seen as the source of type I and type II Fs.These studies have also shown that the formation of tra Fs is a recA-independent process that can occur from the F and Hfr states, that -mediated deletions in pWS200 often demonstrate regional specificity in having endpoints near the ilv operon and that genetic alterations in either replication origin of pWS200 (F oriV, chromosomal oriC) stabilize the replication of this mini-Hfr cointegrate.     

Construction and some properties of packageable plasmid F

Summary A derivative of plasmid F which is packageable in phage coat was constructed using techniques of in vitro recombination. This plasmid is composed of three DNA fragments generated by restriction enzyme EcoRI: a miniF fragment (fragment f5 of F'lac) which is able to replicate autonomously, a DNA fragment from Staphylococcus Plasmid that carries the -lactamase gene, and a portion of guaA (B) transducing phage DNA carrying cohesive ends (cos site) along with almost all the late genes but devoid of all those genes and sites that are needed for replication, regulation, and recombination. The hybrid plasmid has a molecular weight of 2.7×10⁷ daltons, about 84% size of phage genome, and can be packaged in coat when helper phage replicates in the plasmid-carrier cell. The package plasmid and the helper phage particles are separated by CsCl density gradient centrifugation. The replication characteristics of the recombinant plasmid are all those of F including the copy number, incompatibility, and curing with acidine orange. The packaged plasmid is injected into an F^- cell and establishes a plasmid state with normal efficiency. In F⁺ or Hfr cells, the resident F factor hinders this process.     

Conjugal transfer of E. coli F''lac from Erwinia chrysanthemi to Pseudomonas syringae pv. glycinea and the apparent stable incorporation of the plasmid into the pv. glycinea chromosome

Summary The E. coli Flac plasmid was transferred from an Erwinia chrysanthemi Hfr8 donor to a multiply-auxotrophic, rifampicin-resistant Pseudomonas syringae pv. glycinea recipient. Transfer occurred at a frequency of approximately 10^-5/donor. Stable transconjugants which were able to utilize lactose as the sole carbon source after several transfers would not donate the Flac plasmid in detectable frequency to other pv. glycinea or E. coli recipients. The plasmid DNA was shown to be integrated into the pv. glycinea chromosome (Fig. 1).     

Temperature-sensitive mutations affecting the replication of F-prime factors in Escherichia coli K 12

Summary More temperature-sensitive mutants affecting the replication of the F-gal⁺ episome of Escherichia coli K12 have been isolated. Eight of the mutations were located on F itself and three were located on the chromosome.The temperature sensitive F-gal⁺'s have been integrated into the chromosome to produce Hfr strains. These Hfr strains have transfer origins similar to Hfr Cavalli, and all show aberrant excision and transfer of elongated segments of the chromosome including the integrated F-gal to generate long merodiploids.The chromosomal mutations that govern the replication of F have been termed seg (for segregation). Wild-type F-gal⁺ can be integrated into seg cells at 42° C to give Hfrs, in a process analogous to integrative suppression in the formation of Hfrs from cells carrying mutations that are temperature-sensitive for chromosomal DNA replication (dnaA). A curious feature of an Hfr derived from a seg strain is that it also shows F-genote enlargement as well as normal transfer of chromosomal genetic marker. Preliminary transductional mapping data show that the mutation seg-2 is linked to the threonine locus (minute 0).     

Evaluation of an exotic maize population adapted to a locality

Summary An exotic Zea mays L. population (Tuxpeno) was adapted to North Carolina conditions by first introducing genes for adaptability from two North Carolina varieties ([(Jarvis X Indian Chief)Tuxpeno]Tuxpeno) including four generations of intermating, and then selecting for adaptability using maturity as the primary measure. The study evaluated selection for adaptability and the diversity available between adapted Tuxpeno and the local varieties, Jarvis and Indian Chief. Analytical procedures were developed to quantify the diversity between populations and the complementation of local varieties by introduced germ plasms. The analyses utilized the specific effects available from the diallel mating design.Three replicate selections responded similarly under simple recurrent mass selection (1/10) for the earliest disease-free plants initially and additionally for plant types (primarily height) in the final generation. The 1/4 local germ plasm permitted rapid adaptation of Tuxpeno gene pool to local conditions. The adapted Tuxpeno populations yielded similarly to the local populations with an average heterosis for grain yield of 28% when crossed to the local populations used as source of genes for adaptability. The diversity found between adapted Tuxpeno lines and these local varieties based on genes affecting grain yield was 1.5 to 2.5 times that measured between the local varieties (Jarvis and Indian Chief). Diversity lost through intergradation with local material was a reasonable investment. Yield genes introduced from Tuxpeno complemented local gene pools through nonadditive, primarily dominance-associated, gene effects. Reassortment of major gene blocks apparently occurred leading to significant divergence among replicate selections involving both additive-associated and dominance-associated gene effects.Paper No. 6355 of the North Carolina Agri. Res. Ser., Raleigh, NC. Research supported in collaboration with the Rockefeller Foundation and CIMMYT, D.F. (Mexico)     

Inactivation of calcium currents in the soma of spinal ganglia by removing the membrane potential depolarization shift

Kinetic and voltage-dependent characteristics of the inactivating action of incoming calcium currents were investigated in the somatic membrane of rat spinal ganglia neurons using an intracellular dialysis technique. It was shown that the "tail" of low-threshold calcium current could be reliably described by one exponent with a time constant of =1.2–1.8 msec at a repolarization potential of –90 mV. The "tail" of the high-threshold calcium current represented the sum of several exponents. The time constant of the main component which expressed inactivation of the high threshold calcium current was ^h=250–350 µsec. It was also shown that and ^h remained virtually unchanged for repolarization potentials in the subthreshold region; they increase, however, if the repolarizing potential is close to those potentials at which the corresponding component of calcium current is initially activated. A dependence was observed between the levels and ^h and duration of the depolarizing shift. Findings are discussed in the context of a three-tier model of calcium channels.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 17, No. 5, pp. 682–691, September–October, 1985.     

Early and late transfer of F genes by Hfr donors of E. coli K-12

Summary The results of short interrupted matings between an Hfr donor and a recipient strain carrying a temperature-sensitive replication mutant (frp ^–) of Flac demonstrate that the Hfr strain transfers this frp gene of F early in conjugation. This frp gene was also shown to function in the maintenance of mutant F plasmids which appear to be generated from the DNA transferred early in conjugation by Hfr donors. In the course of these experiments, it was further demonstrated that certain Hfr strains which had been described as transferring the tra genes early in fact transfer that region of F late in conjugation.     

Chromosomal location of esterase,peroxidase and phosphoglucomutase isozyme structural genes in cultivated rye (Secale cereale L.)

Summary Isoelectric focusing of esterase (EST), peroxidase (PRX), and phosphoglucomutase (PGM) isozymes in Chinese Spring wheat, Imperial rye and several Chinese Spring/Imperial and Holdfast/King II addition, translocation and substitution lines revealed the chromosomal location of nine Est loci previously described and of one Prx and Pgm locus. Loci Est1, Est2, Est3, Est5, Est6 and Est7 were found on chromosome arm 5RL, Est8 and Est9 on chromosome 6R in Imperial rye, and the Est10 locus on chromosome arm 4RL in Imperial rye and King II rye. A discrepancy was found between the chromosomal location of the Prx locus in Imperial where chromosome 2R was responsible for the expression of the peroxidase enzyme, and King II with chromosome 1R carrying the Prx gene. As a possible explanation, the occurrence of translocation events during the production of wheat/rye aneuploid lines is discussed. The rye Pgm locus could be associated with chromosome 4RS in Imperial and King II rye. Except for the location of Est loci on chromosome 5RL, the results reported in this paper lend further evidence for the assumed homoeology relationships between the chromosomes of Triticinae and for the conservation of gene synteny groups during the evolution of the Triticeae tribe.     

Analysis of QTLs for yield, yield components, and malting quality in a BC3-DH population of spring barley

Advanced backcross (AB)-quantitative trait locus (QTL) analysis has been successfully applied for detecting and transferring QTLs from unadapted germplasm into elite breeding lines in various plant species. Here, we describe the application of a modified AB breeding scheme to spring barley. A BC₃-doubled haploid (DH) population consisting of 181 lines derived from the German spring barley cultivar Brenda (Hordeum vulgare subsp. vulgare) as the recurrent parent and the wild species line HS213 (H. vulgare subsp. spontaneum) as the donor line was evaluated for yield and its components as well as malting quality traits. A set of 60 microsatellite markers was used to genotype the population, and phenotypic data were collected at two locations in Germany in continuous years. Altogether, 25 significant QTLs were detected by single-marker regression analysis and interval mapping. Most positive QTLs originated from the recurrent parent Brenda. A QTL, Qhd2.1, on chromosome 2HS from Brenda explained 18.3% and 20.7% of the phenotypic variation for yield and heading date, respectively. Due to the small percentage of donor-parent genome of 6.25%, the BC₃-DH lines could be directly used for the extraction of near-isogenic lines (NILs) for Qhd2.1. Consequently, it was possible to determine the precise location of the locus hd2.1 within a region of 6.5 cM, using an F₂ population consisting of 234 individuals developed from a cross between an NIL containing a defined donor segment at this locus and Brenda. The location of this QTL was consistent with the presence of a major photoperiod response gene, Ppd-H1, previously reported in this region, which is associated with pleiotropic effects on yield components. In summary, the analysis of a BC₃-DH population in barley provides a compromise between the analysis of QTLs by means of an AB scheme and the generation of defined substitution lines. Several lines carrying defined different donor segments for only one single chromosome or trait in the genetic background of Brenda could be selected for further genetic studies.     

Genetic mapping between Gli-B1 locus and a telomeric C-heterochromatin band in wheat

Summary Using C-banding it has been possible to prove that the bread wheat varieties Holdfast and CapelleDesprez shows an intense band of telomeric heterochromatin on the short arm of chromosome 1B, while the variety Pané-247 presents a very thin band. Gliadin study using pH-acid electrophoresis revealed the existence of differences in the Gli-B1 locus in the three varieties. Analysis of the progeny of the (P x H) x CD hybrid revealed recombination between the heterochromatin C-band and locus Gli-B1, and allowed the genetic distance between the two markers to be calculated as 6.55±3.16 cMorgan. This is the first time the genetic distance from a locus to the chromosome telomere has been directly obtained in wheat. The heterochromatin C-band studied here gives us a cytological marker on chromosome 1B that can be used as a reference point in the localization of other genes.     

Circadian oscillators,cell cycles,and singularities: light perturbations of the free-running rhythm of cell division inEuglena

Summary The free-running circadian rhythm of cell division in the algal flagellate,Euglena gracilis (Z) was perturbed by 3-h light signals of varying intensities imposed at different circadian times (CT). Light pulses within the range of 700 to 7,500 lux were found to yield the same strong (Type 0) phase response curve (PRC) comprising both advance and delaye phase shifts as great as 15 h. Dark signals generated a PRC of reduced amplitude with very little, if any, phase advance being observed. Light perturbations of lower intensity, however, elicited quite different responses if applied at a quite specific circadian time: A 40- to 400-lux pulse given at approximately CT 0 (late subjective night) induced total arrhythmicity, and the culture reverted to asynchronous, exponential growth. Different degrees of arryhtmicity were induced by the same low-intensity perturbations (I ^*) given slightly before or after this sensitive phase point (T ^*), but if imposed at other circadian times, they generated normal type 0 phase resetting. The demonstration of the existence of this critical pulse (T ^*,I ^*) provides further evidence that the cell division cycle ofEuglena (and presumably other microorganisms) is regulated by a circadian oscillator and, in particular, by one having limit cycle dynamics.Abbreviations LL continuous illumination - DD continuous darkness - LD light-dark cycle - LD x, y, light-dark cycle comprisingx h of light andy h of dark - t period of a LD cycle - CO circadian oscillator - CR circadian rhythm - period of a freerunning circadian rhythm in constant conditions (taken here to be the time between onsets of cell division in a population of cells - _R phase marker, or phase reference point (here, the onset of the division burst) - phase of the rhythm - change in phase (phase shift) - new phase attained after phase shift - CT circadian time (CT 0 indicates the phase point of a free-running rhythm that has been normalized to 24 h which corresponds to that occurring at the onset of light in aLD:12, 12 reference cycle) - PRC phase response curve (plot of phase shift engendered by a perturbation as a function of the circadian time of its application) - T ^*,I ^*) coordinates of an annihilating (light) stimulus given at a critical circadian time (T ^*, corresponding to the singularity point) and having a critical strength (I ^*) - CDC cell division cycle - average generation (doubling) time of a cell population - average step-size, or factorial increase in cell titer (plateau to plateau) after a phased division burst Dedicated to Prof. Colin S. Pittendrigh on only his 65th birthday     

Influence of glucose on production and N-sulfation of heparan sulfate in cultured adipocyte cells

Nitric oxide (NO), a reactive nitrogen species, plays an important role in inflammatory lung damage. In the present study, we investigated the role of NO in DNA-binding activity of NF-B in macrophages stimulated with silica or other inflammatory stimulants. Treatment of mouse macrophages (RAW264.7 cells) with a selective inhibitor of inducible nitric oxide synthase (iNOS), L-N⁶-(1-iminoethyl) lysine (L-NIL), or a nonselective iNOS inhibitor, N-nitro-L-arginine methylester (L-NAME), resulted in inhibition of silica-induced nitric oxide production as well as silica-induced NF-B activation. L-NIL also effectively inhibited NF-B activation induced by other inflammatory stimulants, such as lipopolysaccharide (LPS) or muramyl dipeptide (MDP). These inhibitory effects of L-NIL and L-NAME on silica- or LPS-induced NF-B activation were also observed in primary rat alveolar macrophages. Furthermore, NO generating compounds, such as sodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1), caused a dose-dependent increase in NF-B activation, which was positively correlated with the level of NO production. Specific inhibitors of protein tyrosine kinase, such as genistein and AG494, prevented NF-B activation in SNP- or SIN-1 treated cells, suggesting involvement of tyrosine kinase in the NO signaling pathway leading to NF-B activation. In contrast, inhibitors of protein kinase C or A, such as staurosporine or H89, had no inhibitory effect on SIN-1 induced NF-B activation. Metalloporphyrins, such as tetrakis (N-methyl-4-pyridyl) porphyrinato iron (III) (Fe-TMPyP) and Zn-TMPyP which are known to alter NO-dependent activity, markedly inhibited silica- and LPS-induced NF-B activation. The results suggest that NF-B activation in macrophages can be induced under certain conditions by nitric oxide and that nitric oxide produced by phagocytes exposed to inflammatory agents may up-regulate the activation of NF-B.     

Genetic characteristics of two genes for resistance to soybean mosaic virus in PI486355 soybean

Soybean [Glycine max (L.) Merr.] PI486355 is resistant to all the identified strains of soybean mosaic virus (SMV) and possesses two independently inherited resistance genes. To characterize the two genes, PI486355 was crossed with the susceptible cultivars Lee 68 and Essex and with cultivars Ogden and Marshall, which are resistant to SMV-G1 but systemically necrotic to SMV-G7. The F₂ populations and F₂₃ progenies from these crosses were inoculated with SMV-G7 in the greenhouse. The two resistance genes were separated in two F₃₄ lines, LR1 and LR2, derived from Essex x PI486355. F₁ individuals from the crosses of LR1 and LR2 with Lee 68, Ogden, and York were tested with SMV-G7 in the greenhouse; the F₂ populations were tested with SMV-G1 and G7. The results revealed that expression of the gene in LR1 is gene-dosage dependent, with the homozygotes conferring resistance but the heterozygotes showing systemic necrosis to SMV-G7. This gene was shown to be an allele of the Rsv1 locus and was designated as Rsv1-s. It is the only allele identified so far at the Rsv1 locus which confers resistance to SMV-G7. Rsv1-s also confers resistance to SMV-G1 through G4, but results in systemic necrosis with SMV-G5 and G6. The gene in LR2 confers resistance to strains SMV-G1 through G7 and exhibits complete dominance. It appears to be epistatic to genes at the Rsv1 locus, inhibiting the expression of the systemic necrosis conditioned by the Rsv1 alleles. SMV-G7 induced a pin-point necrotic reaction on the inoculated primary leaves in LR1 but not in LR2. The unique genetic features of the two resistance genes from PI486355 will facilitate their proper use and identification in breeding and contribute to a better understanding of the interaction of SMV strains with soybean resistance genes.     

Transfer-deficient cointegrates of Flac and lambda prophage

Summary Twelve transfer-deficient mutants of the plasmid Flac were obtained by insertion of prophage lambda into secondary attachment sites within the transfer region. Insertions into eight different tra genes were identified. These mutations were strongly polar on expression of tra genes previously mapped downstream, and thus confirmed that the genes traA through traD form a single operon. However, some continued expression of traI suggested that this was transcribed in part from a promoter located between traD and tral, and in part from the transfer operon promoter. One insertion early in the transfer operon produced a plasmid-specific tra mutation not complemented by R100-1 or R1-19: this insertion was into a new gene (traY), located before traA as the first member of the transfer operon. Partial tra deletion mutants were obtained as 42° C — survivors from several of the Flac tra:: ED4 plasmids, and their properties are described.     

A difference in UV-sensitivity between genes in the repressed and genes in the de-repressed state

Summary The sensitivity of the capacity for forming galactokinase inE. coli was measured under various conditions. Differences were found between strains W 8 and Hfr 7 () and between these strains and mutants, derived from them, which are partially constitutive for galactokinase. The same behavior as that of the constitutive mutants was also obtained for Hfr 7 () induction of the genes for galactokinase or -galactosidase prior to UV-irradiation. It was concluded that these genes exhibit different sensitivities toward UV, depending on whether they are in the repressed or de-repressed state. Pre-induction effects on UV-sensitivity are observed only in strain Hfr 7 () and only if the latter is lysogenic for phage .With 6 Figures in the Text     

Transposition of IS1-lambdaBIO-IS1 from a bacteriophage lambda derivative carrying the IS1-cat-IS1 transposon (Tn9)

Summary Tn9 is a transposable element in which a gene (cat) determining chloramphenicol resistance is flanked by directly repeated sequences that are homologous to the insertion sequence IS1. We show here that infection of Escherichia coli K12 (under Rec^- Red^- Int^- conditions) with a bio transducing phage carrying Tn9 results in the formation of bio transductants as frequently as cat transductants (about 1 per 10⁶ to 10⁷ infected cells). Most of the bio transductants do not carry cat, just as most of the cat transductants do not carry bio. In spite of the absence of cat, the bio prophage can transpose a second time, from the E. coli chromosome to different sites on an F gal plasmid. Analysis of the structure of the transposed bio element, by restriction nuclease digestion and by electron microscopy, demonstrates that the integrated bio prophage is flanked by directly repeated IS1 elements. We conclude that there is no genetic information for the ability to transpose encoded in the non-repeated portion of Tn9, i.e. that the directly repeated IS1 elements alone are responsible for Tn9 transposition.     

Effects of moisture stress on the multiplication and expansion of cells in leaves of sugar beet

Summary Sugar beets were subjected to moisture stress by decreasing the water potential of the culture solution osmotically with polyethylene glycol by a known amount, , and, alternatively by applying matric potential, , at the plant roots. Lowering the water potential at the root surface less than 200 millibars by either method resulted in significant decreases in the rate of cell multiplication. The final number of cells per leaf at = -372 mb the final was 165% of that at = -473 mb ( = –101 mb); similarly at = –15 mb the final cell number was 198% of that at = –196 mb ( = –181 mb). The mean cell volume of leaves was not significantly affected by these levels of moisture stress.     

Comparison of retroviral p15E-related factors and interferon α in head and neck cancer

Head and neck squamous cell carcinomas (HNscc) produce low-molecular-mass factors (low-M _r factors,M _r25,000), which are antigenically related to the immunosuppressive retroviral transmembrane envelope protein p15E. These P15E-related tumour factors are thought to be responsible for some immunological impairments found in these patients (particularly the defective monocyte chemotaxis). A sequential and functional homology has been reported to exist between a bioactive fragment of interferon (IFN) and the putative immunosuppressive region of retroviral p15E (CKS-17). In this study we investigated (a) a possible functional and structural relationship between p15E and IFN, and (b) the presence of and the relationship between p15E-related low-M _r factors and IFN in HNscc patients. We report the following results. (a) Recombinant human (rhu) IFN was able to inhibit monocyte chemotaxis. (b) The anti-p15E antibodies crossreacted with rhuIFN in a dot-blot technique, however, the anti-IFN antibodies did not crossreact with disrupted murine leukaemia virus (p15E source). (c) Low-M _r factors (n=8–11) prepared from the sera of HNscc patients, which inhibit the monocyte chemotactic responsiveness, could be adsorbed by the anti-p15E antibodies as well as by the anti-IFN antibodies. However, the abilities of the factors to adsorb to the two categories of antibodies (namely, anti-p15E and anti-IFN) did not correlate. (d) Immunohistochemically we found IFN-related epitopes, in almost all HNscc specimens studied (17/18), in locations distinctive from those of p15E-related factors. The anti-IFN antibodies used in this study mainly reacted with basal epithelial cells close to the basal membrane, the prickle and granular cells of the squamous cell carcinomas. The anti-p15E antibodies mainly reacted with corneal layers, the granular and prickle cells, and did not react with basal epithelial cells. Our findings suggest that the immunosuppressive factors produced by HNscc cells are heterogeneous and p15E- and/or IFN-related.     

Genetic analysis of certain in vitro and in vivo parameters in pigeonpea (Cajanus cajan L.)

Summary Studies on callus growth and shoots/cotyledon, using seven different genotypes of pigeonpea and their hybrid progenies, revealed continuous variation for these traits. Hence, the type of gene action influencing in vitro cell proliferation and differentiation has been investigated in a diallel analysis of seven pigeonpea genotypes. Highly significant average heterosis was recorded for callus growth and seed yield/plant. In general, the F₁ hybrids which showed heterosis for callus growth also exceeded their better parent for yield/ plant. Combining ability analysis revealed both additive and non-additive gene effects for callus growth, while number of shoots/cotyledon was mostly governed by non-additive gene effects. The genotype, ICP 7035, was the best general combiner for callus growth and shoot forming capacity of cotyledons. Two cross combinations, 7186×6974 and 7035×T-21, showed maximum SCA effects for callus growth and shoots/cotyledon. Callus dry weight was positively correlated with seed yield/plant and seedling weight. The strong positive association of callus growth with seed yield indicates the possibility of using this system for mass screening and selection of superior hybrids.