Site-specific integration of the phage ΦCTX genome into the Pseudomonas aeruginosa chromosome: characterization of the functional integrase gene located close to and upstream of attP

The site-specific integration of the phage ?CTX genome, which carries the gene for a pore-forming cytotoxin, into the Pseudomonas aeruginosa chromosome was analysed. The 1,167 by integrase gene, int, located immediately upstream of the attachment site, attP, was characterized using plasmid constructs, harbouring the integration functions, and serving as an integration probe in both P. aeruginosa and Escherichia coli. The attP plasmids p1000/p400 in the presence of the int plasmid pIBH and attP-int plasmids pINT/pINTS can be stably integrated into the P. aeruginosa chromosome. Successful recombination between the attP plasmid p1000 and the attB plasmid p5.1, in the presence of the int plasmid pIBH in E. coli HB101 showed that the int gene is active in trans in E. coli. The int gene product was detected as a 43 kDa protein in E. coli maxicells harbouring pINT. Proposed integration arm regions downstream of attP are not necessary for the integration process. pINT and phage ?CTX could be integrated together into P. aeruginosa chromosomal DNA, yielding double integrates.     

The J gene of ?X174: Isolation and characterization of a J gene mutant

Summary The J gene protein of bacteriophage X174 is a component of the mature phage. The previous lack of J gene mutants has prevented an in vivo analysis of J protein functions. A X174 mutant was constructed by inserting an 11 nucleotide sequence into the J gene. This mutant, designated insJ, was viable only in the presence of a wild-type J gene carried on a plasmid that could provide J protein. An analysis of DNA synthesis during insJ mutant infection under non-permissive conditions confirmed that the J protein is not required for viral DNA synthesis.     

Stable integration and expression of heterologous genes in several lactobacilli using an integration vector constructed from the integrase and attP sequences of phage ΦAT3 isolated from Lactobacillus casei ATCC 393

An integration vector capable of stably integrating and maintaining in the chromosomes of several lactobacilli over hundreds of generations has been constructed. The major integration machinery used is based on the ΦAT3 integrase (int) and attP sequences determined previously. A novel core sequence located at the 3′ end of the tRNA^leu gene is identified in Lactobacillus fermentum ATCC 14931 as the integration target by the integration vector though most of such sequences found in other lactobacilli are similar to that determined previously. Due to the lack of an appropriate attB site in Lactococcus lactis MG1363, the integration vector is found to be unable to integrate into the chromosome of the strain. However, such integration can be successfully restored by cotransforming the integration vector with a replicative one harboring both attB and erythromycin resistance sequences into the strain. Furthermore, the integration vector constructed carries a promoter region of placT from the chromosome of Lactobacillus rhamnosus TCELL-1 which is used to express green fluorescence and luminance protein genes in the lactobacilli studied.     

Plasmids carrying cloned fragments of RF DNA from the filamentous phage φLf can be integrated into the host chromosome via site-specific integration and homologous recombination

Different regions of RF DNA from the filamentous bacteriophage phiLf were cloned in Escherichia coli vectors that can not be maintained in Xanthomonas. After introduction into X. campestris pv. campestris 17 (Xc17), most of these constructs were found to integrate into the host chromosome, either by recA-dependent homologous recombination or recA-independent site-specific integration. Mutations in himA, which codes for the alpha-subunit of the Integration Host Factor, does not affect the integration. Integration occurs into a chromosomal region which harbors a copy of a defective phage (4445 bp) that shares a high degree of identity with the phiLf genome. While various parts of the 4445-bp region are susceptible to homologous recombination, site-specific integration requires the attB sequence on the chromosome and the phage attP. The attB shows a high level of sequence identity (22 out of 28 bp) to the dif site required for E. coli Xer site-specific recombination, including the 6-bp central region, and 8/11 identity in both the left XerC-binding arm and the right XerD-binding arm, with the innermost 5 nt of the arms forming a dyad symmetry that is also present in dif. The attP has the same central region and shows 10/11 identity to the dif site in the left arm, but the sequence of the right arm is less conserved than that of attB. The smallest regions still capable of mediating integration are a cloned 72-bp phiLf attP-containing sequence and a 51-bp Xc17 attB-containing sequence, which was reinserted into the Xc17 chromosome after the 4445-bp region had been deleted, indicating that accessory sequences are not necessary and that the integrase required for site-specific integration is neither specified by the 4445-bp Xc17 chromosomal region nor encoded by the phiLf genome.     

A genetic approach to the identification of functional amino acids in protein p6 of Bacillus subtilis phage ?29

Protein p6 of the Bacillus subtilis phage ø29 is essential for in vivo viral DNA replication. This protein activates the initiation of ø29 DNA replication in vitro by forming a multimeric nucleoprotein complex at the replication origins. The N-terminal region of protein p6 is involved in DNA binding, as shown by in vitro studies with p6 proteins altered by deletions or missense mutations. We report on the development of an in vivo functional assay for protein p6. This assay is based on the ability of protein p6-producing B. subtilis non-suppressor (su ^–) cells to support growth of a ø29 sus6 mutant phage. We have used this trans-complementation assay to investigate the effect on in vivo viral DNA synthesis of missense mutations introduced into the protein p6 N-terminal region. The alteration of lysine to alanine at position 2 resulted in a partially functional protein, whereas the replacement of arginine by alanine at position 6 gave rise to an inactive protein. These results indicate that arginine at position 6 is critical for the in vivo activity of protein p6. Our complementation system provides a useful genetic approach for the identification of functionally important amino acids in protein p6.     

Cloning and characterization of the bgxA gene from Erwinia chrysanthemi D1 which encodes a β-glucosidase/xylosidase enzyme

A -glucosidase/xylosidase gene from Erwinia chrysanthemi strain D1 was cloned and sequenced. This gene, named bgxA, encodes a ca. 71 kDa protein product which, following removal of the leader peptide, resulted in a ca. 69 kDa mature protein that accumulated in the periplasmic space of E. chrysanthemi strain D1 and Escherichia coli cells expressing the cloned gene. The protein exhibited both -glucosidase and -xylosidase activities but gave no detectable activity on xylan or carboxymethyl cellulose. The enzyme was classified as a type 3 glycosyl hydrolase, but was unusual in having a truncated B region at the carboxyl-terminus. Several E. chrysanthemi strains isolated from corn produced the glucosidase/xylosidase activity but not those isolated from dicot plants. However, bgxA marker exchange mutants of strain D1 were not detectably altered in virulence on corn leaves.     

The care of young Pi?on Jays (Gymnorhinus cyanocephalus) and their integration into the flock

Summary Piñon Jays live year round in a social flock that may number from 50 to 300 birds. Young birds form into creches immediately after leaving the nest. Begging calls are important in the formation of creches. In these creches young interact with each other and with their parents. This study was designed to 1. gain an understanding of the parent-young relationships, 2. investigate the operation of the creche, 3. determine the relationship of young jays to other cohorts in the flock.Young birds begged and were sometimes fed by foster parents in addition to their own parents. Parent-young recognition, however, is shown to be a strong component of the social system. Young removed from the nest at 20 days of age and transported 1.9 km from the nest-site were recognized by their parents 21 hr after removal. Another brood of young learned to beg from these parents, and were eventually fed by them. In a wire enclosure in a field situation foster feeding was also observed. In both cases, males were more prone to feed foster young than females.Nestlings at 14 days of age are incapable of giving loud begging calls. These young apparently recognize their parents. Parents appear not to recognize young of this age when they are removed from the nest. Location of the nest is sufficient information for locating nestlings, but young, by recognizing their parents' calls can be prepared to receive food rapidly. This may be an important anti-predator devise as food is transferred rapidly and efficiently.Fledged young concealed in paper bags were recognized by their parents, thus proving that vocalizations are an important component of parent-young recognition and may serve to reduce confusion in the creche by enhancing contact between parent and young.Social interactions of young birds among themselves and also with other cohorts were systematically recorded at a feeding station. During the summer when creches roamed as units, young birds engaged in a relatively large number of aggressive interactions among themselves and few interactions with other cohorts. No older cohort consistently dominated the young. In fall and early winter when the entire flock had reformed the aggressiveness of the young birds declined dramatically. All cohorts engaged the young less than expected and only adult males clearly dominated them. During courtship and nesting the yearlings acted aggressively at about the same frequency as older females but less than older males. Most cohorts still engaged yearlings less than expected.Throughout their first year, young birds appear to enjoy a special status in the flock and are deferred to by most older birds. Young birds did not act like subordinate birds in terms of their approach to the feeder or in their temporal pattern of feeding. Evidence suggests that parent-young recognition may remain in affect for longer than one year.The feeding of young from other nests may occur as a means to keep the creche relatively quiet so as not to attract predators or it may result because males occasionally are successful in stealing copulations from females other than their mates. Under these circumstances kin-selection arguments cannot be applied independent of individual selection. Apparent altruistic acts appear to have a low cost to benefit ratio.Colonial nesting and the creching of young probably first evolved among non-related individuals. These acts allow members to forage as a flock, mutually defend nests, and divide up the labor of protection of the young. Inclusive fitness will be further enhanced if these acts benefit relatives, thus a premium should be placed on recruiting kin into the group but not to the total exclusion of strangers.

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Pflege und Gruppenintegration der Jungen beim Nacktschnabelhäher(Gymnorhinus cyanocephalus)

Zusammenfassung Nacktschnabelhäher leben ganzjährig in Gruppen von 50–300 Individuen. Jungvögel schließen sich unmittelbar nach dem Ausfliegen zu Krippen zusammen, für deren Zustandekommen die Bettelrufe eine große Rolle spielen. Innerhalb der Krippen kommt es zu sozialen Kontakten zwischen den Jungvögeln untereinander und zwischen Jungen und Eltern. Die vorliegende Arbeit hatte drei Ziele und sollte zum Verständnis der Eltern-Jungen-Beziehungen beitragen, die Lebensweise der Krippen klären und schließlich die Beziehungen junger Häher zu anderen Untergruppen innerhalb des Schwarmes untersuchen. (Mit der Bezeichnung Untergruppen werden hier die Zusammenschlüsse von Tieren gleichen Alters und gleichen Geschlechts innerhalb der Gesamtgruppe verstanden, z. B. alle einjährigen usw.)Jungvögel betteln außer ihren eigenen Eltern auch andere Altvögel an und werden manchmal von ihnen gefüttert. Trotzdem ist das persönliche Erkennen von Eltern und Jungen ein wichtiger Faktor im Sozialsystem dieser Art. Das geht aus Versuchen hervor, in denen Jungvögel im Alter von 20 Tagen aus dem Nest genommen und über eine Entfernung von 1.9 km verfrachtet wurden. Sie wurden von ihren Eltern dort 21 Stunden später wiedererkannt. Die Jungen einer anderen Brut lernten diese Eltern ebenfalls anzubetteln und wurden schließlich von ihnen gefüttert. Entsprechende Fütterungen durch fremde Altvögel wurden auch an Jungvögeln beobachtet, die in einer Drahtvoliere im Aufenthaltsgebiet der Krippe gehalten wurden. In beiden Fällen hatten männliche Altvögel einen größeren Anteil an diesen Fütterungen als Weibchen.Vierzehntägige Nestlinge verfügen noch nicht über laute Bettelrufe. Sie sind jedoch offenbar bereits in der Lage, ihre Eltern am Ruf zu erkennen. Umgekehrt richten sich die Eltern um diese Zeit offenbar noch ausschließlich nach dem Standort des Nestes, da sie aus dem Nest genommene Junge nicht als ihre eigenen erkennen. Das frühe Erkennungsvermögen der Jungen für ihre Eltern ist als Feindanpassung anzusehen: Die Jungen sind durch das Hören der elterlichen Rufe auf die bevorstehende Fütterung vorbereitet, und die Futterübergabe selbst kann daher sehr rasch und wirksam erfolgen.Nach dem Ausfliegen erkennen — wie Versuche mit in Papiertüten verborgenen Jungvögeln zeigten — auch die Eltern ihre Jungen an der Stimme. Dadurch wird ein gegenseitiger Kontakt innerhalb der Krippe gewährleistet.An einer Futterstelle wurden die sozialen Verhaltensweisen zwischen den Jungvögeln und zwischen diesen und den Mitgliedern anderer Untergruppen beobachtet und quantitativ erfaßt. Während der Sommermonate, wenn die Krippen als soziale Einheiten umherziehen, kommt es zu einer großen Anzahl aggressiver Auseinandersetzungen zwischen den Jungen, während deren Zahl gegenüber anderen Untergruppen gering war. Es gab keine Untergruppe, die der Jungengruppe ständig überlegen war. Im Herbst und Winter, als die gesamte Gruppe wieder geschlossen auftrat, kam es zu einem drastischen Absinken der Aggressivität der Jungvögel. Die übrigen Untergruppen ließen sich mit den Jungen weit weniger ein als erwartet, und nur alte Männchen waren ihnen klar überlegen. Während der Balz- und Brutzeit entsprach dann die Aggressivität der Jungvögel des Vorjahres etwa der der adulten Weibchen, war aber noch geringer als die der Männchen. Die meisten Untergruppen hatten mit den Jungen um diese Zeit immer noch weniger soziale Interaktionen als zu erwarten war.Während ihres ersten Lebensjahres scheinen die Jungvögel innerhalb der Gruppe eine eigene soziale Stellung innezuhaben und werden von den meisten Altvögeln respektiert. Jungvögel verhielten sich daher bei der Annäherung an die Futterstelle und bei der Futteraufnahme auch durchaus nicht wie rangtiefe Altvögel. Aus den Beobachtungen geht hervor, daß das persönliche Erkennen zwischen Eltern und Jungvögeln für länger als ein Jahr anhält. Für die erwähnte Fütterung fremder Jungvögel werden zwei mögliche Gründe diskutiert: Sie kann dazu beitragen, die gesamte Krippe relativ ruhig zu halten und das Anlocken von Raubfeinden zu vermeiden; oder sie kann darauf beruhen, daß Männchen gelegentlich auch mit fremden Weibchen erfolgreich kopulieren. In diesem Fall können individuelle und Verwandtschaftsselektion nicht scharf voneinander getrennt werden. Dadurch haben diese scheinbar altruistischen Verhaltensweisen eine niedrige Kosten-Nutzen-Relation. Hinzu kommt, daß sie wahrscheinlich reziprok auftreten.Die biologische Bedeutung der Sozialstruktur des Nacktschnabelhähers wird diskutiert: Koloniebrüten und Krippenbildungen von Jungvögeln haben sich zunächst wahrscheinlich bei nicht näher miteinander verwandten Individuen entwickelt. Ein solcher Zusammenschluß bietet mehrere Vorteile: Die Tiere können als Gruppe der Nahrungssuche nachgehen, ihre Nester gemeinsam verteidigen und in bezug auf den Schutz der Jungen eine Art Arbeitsteilung entwickeln.Ihre Gesamteignung (inclusive fitness) kann noch weiter erhöht werden, wenn ein solcher Zusammenschluß sich auf miteinander verwandte Tiere bezieht. Den höchsten Selektionsvorteil besitzen daher wahrscheinlich solche Gruppen, die bevorzugt verwandte Individuen aufnehmen, jedoch fremde nicht völlig ausschließen.

     

Coping with inadvertent lysis of Escherichia coli cultures: Strains resistant to lysogeny and infection by the stealthy lysogenic phage Φ80

Phage Φ80 can infect Escherichia coli in a stealthy manner and persist by forming lysogens. Such Φ80 lysogens are fairly common and often go undetected unless the host is grown at temperatures below 37°C. Since low growth temperatures are required for growing temperature-sensitive mutants and often preferred for large-scale applications such as protein production, Φ80-resistant strains would be useful. We report the construction of E. coli strains that cannot be efficiently lysogenized or infected by bacteriophage Φ80. These strains contain combinations of deletions or mutations in the bacterial attachment site for Φ80 integration and/or deletions in the genes required for phage absorption to the host outer membrane. These strains should help contain and prevent Φ80 infection of E. coli cultures in a laboratory or industrial setting.     

Mutagenic specificity of N′-methyl-N′-nitro-N-nitrosoguanidine in the gpt gene on a chromosome of Chinese hamster ovary cells and of Escherichia coli cells

Summary DNA base sequence changes induced by N-methyl-N-nitro-N-nitrosoguanidine (MNNG) mutagenesis have been determined for the Escherichia coli gpt gene stably incorporated in a chromosome of Chinese hamster ovary cells and in the chromosome of both growing and starving E. coli cells, instead of on a plasmid as in most previous studies. In the three cases, nearly all mutations were G: C to A: T transitions, with a 2-to 4-fold higher mutation rate, compared to other sites, at guanines flanked on the 5 side by another guanine. Mutagenic hot spots in these experiments were less prominent than in published results for MNNG mutagenesis of gpt and of other genes. A suggested explanation involves repair of O⁶meG. At low levels of mutagenic products, most are repaired and even small differences in the repair rates leads to large differences in the relative amounts of residual O⁶meG at various sites; in contrast, at high levels of mutagenic products there is little effect of repair on the distribution.Abbreviations MNNG N-methyl-N-nitro-N-nitrosoguanidine - MNU N-methyl-N-nitrosourea - O⁶meG O⁶-methylguanine - N7meG N7-methylguanine - CHO Chinese hamster ovary     

Reproductive isolation in Drosophila: how close are we to untangling the genetics of speciation?

Our understanding of the genetic basis of reproductive isolation in Drosophila has progressed rapidly over the past decade. Details of the genetic structure of hybrid sterility have been revealed and a general consensus has been reached concerning the genetic bases of Haldane's rule. Genetic analyses now reach beyond hybrid sterility and inviability, allowing us to make important comparisons across different traits involved in reproductive isolation. Expansion of genetic studies to include rescue of hybrid incompatibilities has opened the door for more detailed molecular and developmental analyses of reproductive isolation than has ever before been possible.     

Schistosoma japonicum: Cloning, expression and characterization of a gene encoding the α5-subunit of the proteasome

The development of an effective vaccine against the schistosome is thought to be the most desirable means to control schistosomiasis, even though there is an effective means of chemotherapy with praziquantel. A full-length cDNA encoding the Schistosoma japonicum proteasome subunit alpha type 5 protein (SjPSMA5) was first isolated from 18-day-schistosomulum cDNAs. The cDNA had an open reading frame (ORF) of 747 bp and encoded 248 amino acids. Real-time quantitative RT-PCR analysis revealed that SjPSMA5 is up-regulated in 18-day and 32-day schistosomes, and the level of expression in male is around fourfold higher than that in female worms at 42 days. The SjPSMA5 was subcloned into pET28a(+) and expressed as inclusion bodies in Escherichia coli BL21 (DE3) cells. Western blotting showed that the recombinant SjPSMA5 (rSjPSMA5) was immunogenic. After immunization of BALB/c mice with rSjPSMA5, reductions of 23.29% and 35.24% were obtained in the numbers of worms and eggs in the liver, respectively. The levels of specific IgG antibodies and cells were significantly higher (P < 0.01) in the group vaccinated with rSjPSMA5 combined with Seppic 206 adjuvant than in the other groups, as detected by enzyme linked immunosorbent assay (ELISA) and flow cytometry. The study suggested that rSjPSMA5 induced partial immunoprotection against S. japonicum in BALB/c mice, and it could be a potential vaccine candidate against schistosomiasis.     

Ammonia regulation of the Rhizobium meliloti nitrogenase structural and regulatory genes under free-living conditions: Involvement of the fixL gene product?

Summary The expression under microaerobic conditions of the Rhizobium meliloti nifA and consequently the nifHDK genes was found to be negatively regulated by ammonia and nitrate. Assimilation of the ammonia to glutamate and glutamine is not required for this regulation to occur. This indicates that ammonia itself, and not a product of its metabolism, may be regulating nif expression. Unlike the situation in Klebsiella pneumoniae, NtrC is apparently not involved in mediating the ammonia effect on nifA expression in R. meliloti. Neither does the fixK gene product, which is known to regulate nifA in R. meliloti, appear to be involved in mediating the ammonia effect. The regulation of nifA by ammonia is shown to be mediated through the FixL protein. A truncated fixJ gene, the product of which has been shown to induce nifA expression irrespective of the oxygen status of the cell, also circumvented the repressive effect of ammonia on nifA expression. This suggests that the ammonia effect is mediated through the FixLJ regulatory cascade. Interstingly no effect of ammonia on fixK expression was observed.     

δ-Aminolevulinate dehydratase: Induced expression and regional assignment of the human gene to chromosome 9q13»qter

Summary The structural gene encoding human -aminolevulinate dehydratase has been assigned to the long arm of chromosome 9 by somatic cell hybridization techniques using murine erythroleukemia-human fibroblast somatic cell hybrids. Dimethyl sulfoxide induction of erythroid differentiation in these hybrid cells resulted in a 3 to 12-fold increase in the levels of total -aminolevulinate dehydratase. Human -aminolevulinate dehydratase was detected by an immunodiscrimination assay using polyclonal mouse anti-human aminolevulinate dehydratase antibodies. Of four primary hybrid clones, each from an independent fusion, one hybrid line, XX-8, was positive for human -aminolevulinate dehydratase. Examination of 23 secondary, tertiary, and quaternary XX-8 subclones revealed that the expression of the human isozyme segregated with human chromosome 9q, confirming the provisional regional assignment made by classical linkage studies. One positive quaternary clone, XX-8-H21-H7-2, expressed human -aminolevulinate dehydratase activity and contained only human 9q13»qter. In addition, studies of tetiary and quaternary subclones from two series, XX-8-A31 and XX-8-H21-H7, indicated that murine regulatory factors increased the human as well as the murine enzymatic activity following induction of erythroid differentiation.