Ovarian maturation in Leucophaea maderae: Juvenile hormone regulation of thymidine uptake into follicle cell DNA

Our research demonstrates that juvenile hormone (JH I) stimulates thymidine incorporation into ovarian follicle cell DNA in the ovoviviparous cockroach, Leucophaea maderae.A rapid, quantitative method for monitoring ³H-thymidine incorporation into ovarian DNA, in vitro, is described. Cultured ovarian tissue from L. maderae incorporates ³H-thymidine into DNA at a linear rate between 16 and 120 min; analysis of the incorporated label revealed at least 98% of it to be in DNA.Using L. maderae females that had been mated 7 days after adult emergence, we monitored the following biochemical phenomena during the 18–22 day period of terminal oöcyte growth: (1) ³H-thymidine incorporation into ovarian DNA: (2) general protein synthesis in fat body; and (3) specific fat body vitellogenin synthesis.Decapitation of mated females with maturing oöcytes arrested both ovarian DNA synthesis and fat body vitellogenin synthesis. Substantial restoration of both types of synthesis was induced by injection of JH I. The resumption of thymidine incorporation into DNA was localized in the follicular epithelium of the terminal oöcyte.In decapitated virgin females, injection of JH I stimulated oöcyte growth and ³H-thymidine incorporation into ovarian DNA. Dose and time response curves indicate that peak stimulation of ovarian DNA synthesis occurred between 72 and 96 hr after administration of a single optimal dose of 25 μg JH I. The concurrent manifestation of ³H-thymidine uptake into ovarian DNA and activity within the fat body indicates that a similar hormonal mode of action may be operative with respect to both tissue types in virgin females.     

Biochemical and genetic characterization of ChiA,the major enzyme component for the solubilization of chitin by<Emphasis Type="Italic"> Cellulomonas uda</Emphasis>

Cellulomonas uda efficiently solubilized chitinous substrates with a simple chitinase system composed of an endochitinase, designated ChiA, which hydrolyzed insoluble substrates into long-chain chitooligosaccharides, and an as yet uncharacterized exochitinase activity. ChiA, isolated from culture supernatant fluids, was found to be a glycosylated endochitinase with an apparent molecular mass of approximately 70 kDa and pI of 8.5. The gene encoding ChiA was cloned in Escherichia coli and sequenced, revealing an open reading frame of 1,716 bp encoding a 571-amino-acid protein with a predicted molecular mass of 59.2 kDa. The region upstream of chiA included a conserved –35 hexamer flanked by two direct repeats analogous to those found in many Streptomyces chitinase promoters, and thought to function as binding sequences for regulatory proteins. Analysis of the deduced amino acid sequence showed a modular protein consisting of a signal peptide at its N terminus, a family 2 carbohydrate-binding module (CBM2) that was closely related to the substrate-binding domains of glycosyl hydrolases from distantly related bacteria, and a family 18 glycosyl hydrolase catalytic module related to Streptomyces chitinases. In contrast to the fibronectin type III domains of Streptomyces chitinases, the linker region between modules in ChiA consisted of a long proline- and threonine-rich module, thought to contribute to the glycosylation and flexibility of the mature protein.Abbreviations CBM Carbohydrate-binding module - P-T Proline- and threonine-rich domain - Fn3 Type III repetitive sequences of fibronectin domain - PKD Polycystic kidney disease I domain     

Connecting replication and recombination

Replication forks frequently break and must be repaired by recombination. A reconstituted reaction now allows the factors that coordinate conversion from a recombination intermediate back to a replication fork to be defined. The PriA protein plays a key role in this control.     

Resolution of parvovirus dimer junctions proceeds through a novel heterocruciform intermediate

The minute virus of mice initiator protein, NS1, excises new copies of the left viral telomere in a single sequence orientation, dubbed flip, during resolution of the junction between monomer genomes in palindromic dimer intermediate duplexes. We examined this reaction in vitro using both (32)P-end-labeled linear substrates and similar unlabeled templates labeled by incorporation of [alpha-(32)P]TTP during the synthesis. The observed products suggest a resolution model that explains conservation of the hairpin sequence and in which a novel heterocruciform intermediate plays a crucial role. In vitro, NS1 initiates two replication pathways from OriL(TC), the single active origin embedded in one arm of the dimer junction. NS1-mediated nicking liberates a base-paired 3' nucleotide to prime DNA synthesis and, in a reaction we call "read-through synthesis," forks established while the substrate is a linear duplex synthesize DNA in the flop orientation, leading to DNA amplification but not to junction resolution. Nicking leaves NS1 covalently attached to the 5' end of the DNA, where it can serve as a 3'-to-5' helicase, unwinding the NS1-associated strand. In the second pathway, resolution substrates are created when such unwinding induces the palindrome to reconfigure into a cruciform prior to fork assembly. New forks can then synthesize DNA in the flip orientation, copying one cruciform arm and creating a heterocruciform intermediate. Resolution proceeds via hairpin transfer in the extended arm of the heterocruciform, which releases one covalently closed duplex telomere and a partially single-stranded junction intermediate. We suggest that the latter intermediate is finally resolved via an NS1-induced single-strand nick at the otherwise inactive origin, OriL(GAA).     

Effects of nicotine on target biting and resident-intruder attack

The effects of acute administration of nicotine on target biting (defensive) and resident-intruder (offensive) attack of male mice were assessed. In the target biting procedure confined mice received tail shock on a fixed time, 2-min schedule. Under baseline conditions, biting attack directed toward an inanimate target occurred at three distinct rates. A high target biting rate (13.5 +/- 3.8 bites/15 sec) followed shock delivery, an intermediate biting rate (9.6 +/- 4.1 bites/15 sec) occurred during the inter-shock interval, and a low biting rate (1.0 +/- 0.5 bites/15 sec) occurred during a tone stimulus which signalled the impending shock. Nicotine (administered IP, 15 min presession) reduced post-shock and inter-shock interval target biting in a dose-dependent manner (ED50 values estimated at 0.13 and 0.14 mg/kg, respectively) but exerted more variable effects on target biting during the tone. In the resident-intruder paradigm the same mice were exposed to an intruder introduced into its home cage for a 10-min test session. Under baseline conditions, residents directed 20 +/- 3.2 biting attacks toward the intruder during the session with an average latency of 89 +/- 40 sec to the first attack. Nicotine caused a dose-dependent decrease in this attack behavior (ED50 values estimated to be 0.48 and 0.49 mg/kg, respectively). These observations are interpreted to indicate that nicotine has an increased potency at reducing "defensive" aggression.     

Neil3 and NEIL1 DNA Glycosylases Remove Oxidative Damages from Quadruplex DNA and Exhibit Preferences for Lesions in the Telomeric Sequence Context

The telomeric DNA of vertebrates consists of d(TTAGGG)_n tandem repeats, which can form quadruplex DNA structures in vitro and likely in vivo. Despite the fact that the G-rich telomeric DNA is susceptible to oxidation, few biochemical studies of base excision repair in telomeric DNA and quadruplex structures have been done. Here, we show that telomeric DNA containing thymine glycol (Tg), 8-oxo-7,8-dihydroguanine (8-oxoG), guanidinohydantoin (Gh), or spiroiminodihydantoin (Sp) can form quadruplex DNA structures in vitro. We have tested the base excision activities of five mammalian DNA glycosylases (NEIL1, NEIL2, mNeil3, NTH1, and OGG1) on these lesion-containing quadruplex substrates and found that only mNeil3 had excision activity on Tg in quadruplex DNA and that the glycosylase exhibited a strong preference for Tg in the telomeric sequence context. Although Sp and Gh in quadruplex DNA were good substrates for mNeil3 and NEIL1, none of the glycosylases had activity on quadruplex DNA containing 8-oxoG. In addition, NEIL1 but not mNeil3 showed enhanced glycosylase activity on Gh in the telomeric sequence context. These data suggest that one role for Neil3 and NEIL1 is to repair DNA base damages in telomeres in vivo and that Neil3 and Neil1 may function in quadruplex-mediated cellular events, such as gene regulation via removal of damaged bases from quadruplex DNA.     

Comparative genetic mapping reveals synteny and collinearity between the American cranberry and diploid blueberry genomes

Cranberry and blueberry are closely related and recently domesticated fruit crops in the genus Vaccinium. Both have a presumed American origin and likely evolved from a common ancestor; however, details of their adaptive radiation and the extent of their genomic divergence remains little understood. To better understand their evolutionary and genomic relationships, a set of 323 cross-transferable simple sequence repeat (SSR) markers were identified, added to existing marker datasets, and used to construct linkage maps for cranberry (582 SSRs) and an interspecific diploid blueberry population (V. darrowii x V. corymbosum) x V. corymbosum (409 markers, densest blueberry SSR map currently available). The maps allowed for the first comparative genetic mapping study in Vaccinium, and revealed a surprisingly high degree of macro-synteny and collinearity between the cranberry and blueberry genomes. Approximately 93% of the blueberry linkage map was collinear with cranberry, while the remaining 7% (66.3 cM) was spread across 15 non-collinear regions detected in eight of the 12 linkage groups. These observations suggest that large-scale genome differentiation between the cranberry and blueberry genomes has not occurred during their evolution, and that sequence information will be highly transferable between the species in future genetic research and breeding. Finally, the set of 323 cross-transferable SSRs and linkage maps they were used to construct can serve as a shared resource for the Vaccinium research community, enabling additional comparative mapping studies, the identification and transfer of quantitative trait loci and candidate genes between species, and future exploration of evolutionary relationships in Vaccinium.     

Sensitive rapid detection method for viable bacterial cells

A rapid sensitive method for the detection of viable bacterial cells is described in which P³² as inorganic orthophosphate is used to label the cells. Factors affecting the uptake of P³² by cells as well as the sensitivity of the method have been explored with suspensions of Aerobacter aerogenes. The uptake of P³²O₄ is dependent on several factors. Of various incubation media tested, one composed of 0.005 m KCl, 0.002 m MgSO₄ and 10 mg/ml of glucose was found to best stimulate the uptake of the tracer. Incubation time and temperature and level of isotope and of unlabeled P also affected uptake. Labeled cells were collected on a membrane filter for measurement of radioactivity. Under optimal conditions, as few as 23 viable cells per milliliter were detected in 1 hr with 95% confidence.     

Structural basis for the lack of opposite base specificity of Clostridium acetobutylicum 8-oxoguanine DNA glycosylase

7,8-Dihydro-8-oxoguanine (8-oxoG) is the major oxidative product of guanine and the most prevalent base lesion observed in DNA molecules. Because 8-oxoG has the capability to form a Hoogsteen pair with adenine (8-oxoG:A) in addition to a normal Watson–Crick pair with cytosine (8-oxoG:C), this lesion can lead to a G:C → T:A transversion after replication. However, 8-oxoG is recognized and excised by the 8-oxoguanine DNA glycosylase (Ogg) of the base excision repair pathway. Members of the Ogg1 family usually display a strong preference for a C opposite the lesion. In contrast, the atypical Ogg1 from Clostridium actetobutylicum (CacOgg) can excise 8-oxoG when paired with either one of the four bases, albeit with a preference for C and A. Here we describe the first high-resolution crystal structures of CacOgg in complex with duplex DNA containing the 8-oxoG lesion paired to cytosine and to adenine. A structural comparison with human OGG1 provides a rationale for the lack of opposite base specificity displayed by the bacterial Ogg.