Fungal diversity in maritime Antarctic soils determined using a combination of culture isolation, molecular fingerprinting and cloning techniques

The diversity and phylogenetic relationships of fungi obtained from Antarctic soils were analysed using molecular techniques. Direct extraction of soil community DNA from two locations, Fossil Bluff (FB) and Jane Col (JC), was supplemented with isolation studies. Nucleic acids from both the community DNA and the colony extracts were PCR amplified using primers specific for the 18S rRNA gene (18S rDNA). Amplicons were separated in denaturant gels (DGGE) or following endonuclease digestion (ARDRA). Clones presenting unique ARDRA banding patterns and unique DGGE bands were sequenced. Comparison of the experimental sequences from the different techniques employed with those held online resulted in the repeated recovery of a limited range of related organisms indicating low species diversity of microfungi in these soils. A total of 102 fungal sequences were obtained from FB (37 sequences) and JC (65 sequences) that together were distributed among the Basidiomycota (48 sequences), Ascomycota (48 sequences) and Zygomycota (6 sequences). Sequences of the latter were only recovered from the JC soils. Phylogenetic comparisons of the experimental sequences with those held online have shown high rRNA gene relatedness with those obtained from other, less extreme, environments.     

Ras-independent activation of the Raf/MEK/ERK pathway upon calcium-induced differentiation of keratinocytes

MAPKs are crucially involved in the regulation of growth and differentiation of a variety of cells. To elucidate the role of MAPKs in keratinocyte differentiation, activation of ERK, JNK, and p38 in response to stimulation with extracellular calcium was analyzed. We provide evidence that calcium-induced differentiation of keratinocytes is associated with rapid and transient activation of the Raf/MEK/ERK pathway. Stimulation of keratinocytes with extracellular calcium resulted in activation of Raf isozymes and their downstream effector ERK within 10-15 min, but did not increase JNK or p38 activity. Calcium-induced ERK activation differed in kinetics from mitogenic ERK activation by epidermal growth factor and could be modulated by alterations of intracellular calcium levels. Interestingly, calcium stimulation led to down-regulation of Ras activity at the same time that ERK activation was initiated. Expression of a dominant-negative mutant of Ras also did not significantly impair calcium-induced ERK activation, indicating that calcium-mediated ERK activation does not require active Ras. Despite the transient nature of ERK activation, calcium-induced expression of the cyclin-dependent kinase inhibitor p21/Cip1 and the differentiation marker involucrin was sensitive to MEK inhibition, which suggests a role for the Raf/MEK/ERK pathway in early stages of keratinocyte differentiation.     

Follicular dynamics prior to and during superovulation in heifers

The present ultrasonographic study examined the relationship between certain follicular parameters and the superovulatory response in gonadotropin-stimulated heifers. Thirty heifers received a total of 35 mg FSH twice daily for 4 d and 0.75 mg cloprostenol were given to induce luteolysis and estrus at 72 h after the initial FSH injection. Transrectal ultrasonography was performed once daily from 1 or 2 d before the initial FSH injection and until the day of estrus. The number of small (2 to 4 mm), medium (5 to 9 mm), and large (>/=10 mm) size follicles as well as the diameter of the large follicles were recorded. Embryos were recovered non-surgically 6 or 7 d after estrus, and the number of corpora lutea was determined by palpation per rectum. Heifers with >2 or 0.05). The number of large follicles and the sum of medium and large follicles were positively correlated (r=0.43 and r=0.54, respectively; P<0.05) with the number of corpora lutea palpated on the day of embryo recovery (6 to 7 d after estrus). In conclusion, there was an effect of the day relative to initiation of FSH treatment on all follicular categories in heifers responding positively to superovulation, and there was no effect of side (left or right ovary) or of corpus luteum diameter (ipsilateral or contralateral).     

DNA loops generate intracentromere tension in mitosis

The centromere is the DNA locus that dictates kinetochore formation and is visibly apparent as heterochromatin that bridges sister kinetochores in metaphase. Sister centromeres are compacted and held together by cohesin, condensin, and topoisomerase-mediated entanglements until all sister chromosomes bi-orient along the spindle apparatus. The establishment of tension between sister chromatids is essential for quenching a checkpoint kinase signal generated from kinetochores lacking microtubule attachment or tension. How the centromere chromatin spring is organized and functions as a tensiometer is largely unexplored. We have discovered that centromere chromatin loops generate an extensional/poleward force sufficient to release nucleosomes proximal to the spindle axis. This study describes how the physical consequences of DNA looping directly underlie the biological mechanism for sister centromere separation and the spring-like properties of the centromere in mitosis.     

Analysis of a repetitive DNA family of two closely related chironomids,Chironomus thummi thummi andCh. thummi piger (Diptera) by density-gradient centrifugation,melting analysis and restriction analysis

The DNAs of the two subspecies ofChironomus thummi, Ch. th. thummi andCh. th. piger, were investigated by CsCl density-gradient centrifugation, melting analysis and restriction analysis including Southern hybridization with AT-rich, highly repetitive DNA sequences. The melting analysis of density-fractionatedCh. th. thummi andCh. th. piger DNA has shown that thethummi DNA contains an early melting DNA fraction, which is enriched in the light fractions of the density gradient. The DNA fraction is also present inpiger DNA though in lower concentration. Restriction and Southern analysis of density fractionatedthummi andpiger DNA has revealed that there are two tandemly-repetitive DNA-sequence families that hybridize with this AT-rich, early melting DNA fraction. One sequence is characterized by anHae-III site and a basic repeat length of 130 ± 15 bp and the other by aCla-1 restriction site and a basic repeat length of 120 ± 4 bp. These sequences are present in much higher concentrations in the genome ofCh. th. thummi when compared toCh. th. piger, and are hence correlated to the higher DNA content of theCh. th. thummi genome.     

A third major locus for autosomal dominant hypercholesterolemia maps to 1p34.1-p32

Autosomal dominant hypercholesterolemia (ADH), one of the most frequent hereditary disorders, is characterized by an isolated elevation of LDL particles that leads to premature mortality from cardiovascular complications. It is generally assumed that mutations in the LDLR and APOB genes account for ADH. We identified one large French pedigree (HC2) and 12 additional white families with ADH in which we excluded linkage to the LDLR and APOB, implicating a new locus we named "FH3." A LOD score of 3.13 at a recombination fraction of 0 was obtained at markers D1S2892 and D1S2722. We localized the FH3 locus to a 9-cM interval at 1p34.1-p32. We tested four regional markers in another set of 12 ADH families. Positive LOD scores were obtained in three pedigrees, whereas linkage was excluded in the others. Heterogeneity tests indicated linkage to FH3 in approximately 27% of these non-LDLR/non-APOB ADH families and implied a fourth locus. Radiation hybrid mapping located four candidate genes at 1p34.1-p32, outside the critical region, showing no identity with FH3. Our results show that ADH is genetically more heterogeneous than conventionally accepted.     

Storage and formulation of the entomopathogenicnematodes Heterorhabditis indica and H. bacteriophora

Successful control of insect pests through theapplication of entomopathogenic nematode dauerjuveniles of H. bacteriophora and H.indica can only be achieved when the nematodematerial reaches the end user in good condition.Storage and formulation techniques must provideoptimum conditions to guarantee a maximum survival andinfectivity of the nematodes. Nematode survival wastested at temperatures ranging between 5–25 °C.A maximum survival of H. indica was achieved at15 °C and the highest mortality at 5 °C.H. bacteriophora survived best at 7.5 °Cand least at 25 °C. An increase of the saltconcentration had positive effects on dauer juvenilesurvival in aqueous suspensions. Low pH between 6 and4 reduced the bacterial growth and prolonged survivalof stored dauer juveniles. Of the organic acidsascorbic, benzoic, citric and sorbic acid, onlyascorbic acid had a positive effect on H. indicasurvival. Extracts of the dried spice plants cinnamon,cloves, rosemary and oregano were tested. Enhancementof H. indica survival was recorded for cinnamonand cloves. Survival and infectivity of nematodesstored in attapulgite and bentonite clays and spongewere recorded over several weeks at different storagetemperatures. Infectivity was not influenced by thedifferent formulation materials. When stored insponge at 25 °C nematodes survived less than 1week and the formulation in clay could only prolongthis period for another week. At 5 °C thesurvival of H. bacteriophora in sponge wassuperior to that in clay, whereas H. indicasurvived less well in sponge than in clay at15 °C. Storage in aerated water at 5 °Cfor H. bacteriophora and at 15 °C for H. indica resulted in the lowest mortality. Forstorage at controlled conditions (temperature, pH andosmolarity), aerated water is superior to all othermethods tested and the addition of preservatives willincrease survival.     

Localization of c-ERB A proteins in rat liver using monoclonal antibodies

Monoclonal antibodies were raised against the nuclear thyroid hormone receptors encoded by c-ERB A genes and against a purified nuclear receptor fraction. These antibodies recognize the c-ERB A protein in nuclear extracts from rat liver and are able to compete with thyroid hormone in Scatchard analyses. In sections of rat liver they react with all the hepatocyte nuclei as well as with the cells of the hepatic bile ducts. Comparison with another putative T3 receptor antibody, described previously, showed that distinct 57 kD proteins with a different cellular distribution were recognized.     

Artificial selection on ant female caste ratio uncovers a link between female‐biased sex ratios and infection by Wolbachia endosymbionts

Social insect sex and caste ratios are well‐studied targets of evolutionary conflicts, but the heritable factors affecting these traits remain unknown. To elucidate these factors, we carried out a short‐term artificial selection study on female caste ratio in the ant Monomorium pharaonis. Across three generations of bidirectional selection, we observed no response for caste ratio, but sex ratios rapidly became more female‐biased in the two replicate high selection lines and less female‐biased in the two replicate low selection lines. We hypothesized that this rapid divergence for sex ratio was caused by changes in the frequency of infection by the heritable bacterial endosymbiont Wolbachia, because the initial breeding stock varied for Wolbachia infection, and Wolbachia is known to cause female‐biased sex ratios in other insects. Consistent with this hypothesis, the proportions of Wolbachia‐infected colonies in the selection lines changed rapidly, mirroring the sex ratio changes. Moreover, the estimated effect of Wolbachia on sex ratio (~13% female bias) was similar in colonies before and during artificial selection, indicating that this Wolbachia effect is likely independent of the effects of artificial selection on other heritable factors. Our study provides evidence for the first case of endosymbiont sex ratio manipulation in a social insect.     

Complete secretion of activable bovine prochymosin by genetically engineered L forms of Proteus mirabilis.

To circumvent problems encountered in the synthesis of active chymosin in a number of bacteria and fungi, a recombinant DNA L-form expression system that directed the complete secretion of fully activable prochymosin into the extracellular culture medium was developed. The expression plasmid constructions involved the in-frame fusion of prochymosin cDNA minus codons 1 to 4 to streptococcal pyrogenic exotoxin type A gene (speA') sequences, including the speA promoter, ribosomal binding site, and signal sequence and five codons of mature SpeA. Secretion of fusion prochymosin enzymatically and immunologically indistinguishable from bovine prochymosin was achieved after transformation of two stable protoplast type L-form strains derived from Proteus mirabilis. The secreted proenzyme was converted by autocatalytic processing to chymosin showing milk-clotting activity. In controlled laboratory fermentation processes, a maximum specific rate of activable prochymosin synthesis of 0.57 x 10(-3)/h was determined from the time courses of biomass dry weight and product formation. Yields as high as 40 +/- 10 micrograms/ml were obtained in the cell-free culture fluid of strain L99 carrying a naturally altered expression plasmid of increased segregational stability. The expression-secretion system described may be generally useful for production of recombinant mammalian proteins synthesized intracellularly as aberrantly folded insoluble aggregates.