Sexual dimorphism and species differences in the neurophysiology and morphology of the acoustic communication system of two neotropical hylids

We examined auditory tuning and the morphology of the anatomical structures underlying acoustic communication in female Hyla microcephala and H. ebraccata and compared our findings to data from a previous study (Wilczynski et al. 1993) in which we showed species differences in the traits that in males relate to differences in the species-typical calls. Female species differences in the best excitatory frequency (BEF) of the basilar papilla (BP) were similar to the differences seen in males, and females had a significantly lower BEF in H. ebraccata, but not H. microcephala. In both species, females had lower BP thresholds. Snout-vent length, head width, and tympanic membrane diameters were sexually dimorphic in both species and larger in females, whereas laryngeal components were sexually dimorphic and larger in males. Middle and inner ear volumes were not sexually dimorphic. Despite the significant species differences in laryngeal morphology seen in males, female larynges are not significantly different. Furthermore, the interaction of species and sex differences resulted in significantly different degrees of sex dimorphism in the species, particularly for the larynx, which is more sexually dimorphic in H. microcephala, and measures of body size, which are more dimorphic in H. ebraccata. Accepted: 6 December 1996     

Screening and identification of yeast sequences that cause growth inhibition when overexpressed

To isolate genes that negatively regulate cell growth, we constructed a galactose-inducible expression library with partially digested Saccharomyces cerevisiae genomic DNA fragments inserted downstream of the GAL10 promoter. In all, 240 000 yeast transformants were screened for lethality on galactose medium. From 17 such transformants identified, 16 nonoverlapping DNA sequences were obtained. Restriction mapping and determination of DNA sequences adjacent to the GAL10 promoter indicated that the inserts encoded part or all of the URA2, RBP1, TPK3, SAC7, BOI1, and BNI1 genes, and also open reading frames (ORFs) from chromosomes IV, V, IX, XI, and XIII. Some of the identified sequences lacked the amino-terminal sequences of the ORFs, suggesting that truncated forms of the proteins might be necessary for growth inhibition. The sequence of the pGA108 insert was highly homologous to the telomeric X-element and contained an ARS consensus sequence, suggesting a possible growth inhibitory effect of an RNA molecule. Overexpression of the BNI1ΔN and BOI1ΔN genes, which lacked amino-terminal sequences, was associated with phenotypes similar to those of mutants defective in bud formation. Overexpression of the GIN4 and GIN12 sequences induced elongated buds and a G2/M arrest-like phenotype, respectively. The phenotypes induced by the overexpression of our cloned sequences could result from either a dominant-positive or a dominant-negative effect and, unexpectedly, in one case from an effect of an RNA. Received: 3 June 1996 / Accepted: 1 October 1996     

Specificity of DNA Methylation Changes During Fungal Dimorphism and Its Relationship to Polyamines

We utilized our modification of the amplified fragment length polymorphism technique for the determination of changes occurring in the DNA methylation patterns during the dimorphic transition of the fungi Mucor rouxii, Yarrowia lipolytica, and Ustilago maydis. To determine the specificity of differential methylation in regards to dimorphism, we obtained the yeast-like form of the three fungi under conditions that induced mycelial growth, by addition of 1,4-diaminobutanone (DAB), an inhibitor of ornithine decarboxylase in the case of M. rouxii and Y. lipolytica. In an odc null mutant of U. maydis, repression of the dimorphic transition was brought about by limitation in the amounts of exogenous putrescine. Yeasts from the three fungi thus obtained conserved a significant number of the differential DNA fragments with the methylation pattern displayed by normal yeasts, indicating their true correlation with dimorphism. Our results also confirm a role of polyamines in differential DNA methylation and fungal dimorphic transition.     

Allelic variation at a hypervariable compound microsatellite locus in the ascomycete Ascochyta rabiei

The genome of the fungal chickpea pathogen Ascochyta rabiei was screened for polymorphisms by microsatellite-primed PCR. While ethidium-bromide staining of electrophoretically separated amplification products showed only limited polymorphism among 24 Tunisian A. rabiei isolates, Southern hybridization of purified PCR fragments to restriction digests of fungal DNA revealed polymorphic DNA fingerprints. One particular probe that gave rise to a hypervariable single-locus hybridization signal was cloned from the Syrian isolate AA6 and sequenced. It contained a large compound microsatellite harbouring the penta- and decameric repeat units (CATTT)_n, (CATTA)_n, (CATATCATTT)_n and (TATTT)_n. We call this locus ArMS1 (Ascochyta rabiei microsatellite 1). Unique flanking sequences were used to design primer pairs for locus- specific microsatellite amplification and direct sequencing of additional ArMS1 alleles from Tunisian and Pakistani isolates. A high level of sequence variation was observed, suggesting that multiple mutational mechanisms have contributed to polymorphism. Hybridization and PCR analyses were performed on the parents and 62 monoascosporic F₁ progeny derived from a cross between two different mating types of the fungus. Progeny alleles could be traced back to the parents, with one notable exception, where a longer than expected fragment was observed. Direct sequencing of this new length allele revealed an alteration in the copy number of the TATTT repeat [(TATTT)₅₃ to (TATTT)₆₅], while the remainder of the sequence was unchanged. Received: 11 March 1997 / Accepted: 21 June 1997     

Artificial circularization of the chromosome with concomitant deletion of its terminal inverted repeats enhances genetic instability and genome rearrangement in Streptomyces lividans

The unstable linear chromosome of Streptomyces lividans was circularized by homologous recombination and its terminal inverted repeats deleted. Strains with circularized chromosomes showed no obvious phenotypic disadvantages compared to the wild type. However, they segregated about 20 times more chloramphenicol-sensitive mutants than the wild type (24.3% vs. 1.4%), due to a higher incidence of large deletions. In addition, in all circularized chromosomes amplification of 30–60 kb fragments was observed at the new chromosomal junction, to levels of approximately 10 copies per chromosome. Arginine auxotrophs that arose spontaneously among the progeny of strains with a circularized chromosome showed high-copy-number amplification of the DNA element AUD1, as also seen in mutants of the wild type. These observations demonstrate that the circular form of the Streptomyces chromosome is more unstable than the linear one. Received: 28 July 1996 / Accepted: 18 November 1996     

Characterization of a novel DNA damage-inducible gene of Saccharomyces cerevisiae , DIN7 , which is a structural homolog of the RAD2 and RAD27 DNA repair genes

A number of DNA damage-inducible genes (DIN) have been identified in Saccharomyces cerevisiae. In the present study we describe isolation of a novel gene, Din7, the expression of which is induced by exposure of cells to UV light, MMS (methyl methanesulfonate) or HU (hydoxyurea). The DNA sequence of DIN7 was determined. By comparison of the predicted Din7 amino acid sequence with those in databases we found that it belongs to a family of proteins which includes S. cerevisiae Rad2 and its Schizosaccharomyces pombe and human homologs Rad13 and XPGC; S. cerevisiae Rad27 and its S. pombe homolog Rad2, and S. pombe Exo I. All these proteins are endowed with DNA nuclease activity and are known to play an important function in DNA repair. The strongest homology to Din7 was found with the Dhs1 protein of S.␣cerevisiae, the function of which is essentially unknown. The expression of the DIN7 gene was studied in detail using a DIN7-lacZ fusion integrated into a chromosome. We show that the expression level of DIN7 rises during meiosis at a time nearly coincident with commitment to recombination. No inducibility of DIN7 was found after treatment with DNA-damaging agents of cells bearing the rad53-21 mutation. Surprisingly, a high basal level of DIN7 expression was found in strains in which the DUN1 gene was inactivated by transposon insertion. We suggest that a form of Dun1 may be a negative regulator of the DIN7 gene expression. Received: 30 May 1996 / Accepted: 26 September 1996     

The regulatory protein MucR binds to a short DNA region located upstream of the muc R coding region in Rhizobium meliloti

The Rhizobium meliloti MucR protein is known to regulate the biosynthesis of the two exopolysaccharides, succinoglycan and galactoglucan. The mucR gene was successfully overexpressed in Escherichia coli BL21 cells by heat shock induction using a two-plasmid system. Cell extracts of the production strain contained about 20% of a polypeptide of 17 kDa apparent molecular mass, corresponding to the size expected for MucR. As shown by an electrophoretic mobility shift assay, these extracts were active in the specific retardation of a 219-bp DNA fragment including 134-bp of the non-coding region upstream of the mucR gene. Primer extension analysis showed that this DNA fragment was located within the transcribed region upstream of the mucR gene. Competition experiments revealed that a 44-bp sequence present within the 134-bp upstream of the mucR gene contained the MucR binding site. Although binding of MucR to this site exhibited a moderate dissociation constant of M, the reaction was highly specific since fragments containing binding sites for the homologous Ros protein from Agrobacterium tumefaciens were not able to compete for MucR binding. Received: 9 October 1996 / Accepted: 20 December 1996     

Flipper, a mobile Fot1-like transposable element in Botrytis cinerea

A transposable element, Flipper, was isolated from the phytopathogenic fungus Botrytis cinerea. The element was identified as an insertion sequence within the coding region of the nitrate reductase gene. The Flipper sequence is 1842 bp long with perfect inverted terminal repeats (ITRs) of 48 bp and an open reading frame (ORF) of 533 amino acids, potentially encoding for a transposase; the element is flanked by the dinucleotide TA. The encoded protein is very similar to the putative transposases of three elements from other phytopathogenic fungi, Fot1 from Fusarium oxysporum, and Pot2 and MGR586 from Magnaporthe grisea. The number of Flipper elements in strains of B. cinerea varied from 0 to 20 copies per genome. Analysis of the descendants of one cross showed that the segregation ratio of Flipper elements was 2:2 and that the copies were not linked. Received: 4 December 1996 / Accepted: 21 January 1997     

A wat1 mutant of fission yeast is defective in cell morphology

The organization of the actin cytoskeleton plays an integral role in cell morphogenesis of all eukaryotes. We have isolated a temperature-sensitive mutant in Schizosaccharomyces pombe, wat1-1, in which acting patches are delocalized, resulting in an elliptically shaped cell phenotype. Molecular cloning and DNA sequencing of wat1 ⁺ showed that the gene encodes a 314 residue protein containing WD-40 repeats. Cells lacking wat1 ⁺ are slow growing but viable at 25° C and temperature-sensitive for growth above 33° C. At restrictive temperature, wat1-d strains are phenotypically indistinguishable from wat1-1. When combined with a deletion for the wat1 ⁺ gene, cdc mutants failed to elongate at restrictive temperature and exhibited alterations in actin patch localization. This analysis suggests that wat1 ⁺ is required directly or indirectly for polarized cell growth in S. pombe. Wat1p and a functional, epitope-tagged, version of Wat1p can be overproduced without inducing alterations in cell morphology. Received: 18 September 1996 / Accepted: 22 October 1996     

Somatic variation during long term subculturing of plant cells caused by insertion of a transposable element in a phenylalanine ammonia-lyase (PAL) gene

We have identified a new En/Spm-like transposable element, Tdc1, in the 5′ flanking region of a phenylalanine ammonia-lyase gene (gDcPAL1) that is normally induced by transferring cells of carrot suspension cultures to fresh liquid medium (transfer or dilution effect). The initial integration into gDcPAL1 occurred more than 4 years after culture initiation. Tdc1 was first detected in gDcPAL1 genomic clones of a genomic library made from cells of the same cultured cell line 7 years after its initiation and thus following repeated subculturing. Twelve years after initiation, about 5–10% of the cells had Tdc1 inserted into the gDcPAL1 gene, indicating that Tdc1 insertion into gDcPAL1 occurred in one (or more) cell(s) during the first 4–7 years of subculturing. These mutant cells did not disappear during numerous passages; instead the proportion of cells having this Tdc1 inserted into gDcPAL1 has been increasing over the last 5 years. The promoter activity and the inducibility by transfer/dilution of the gDcPAL1 gene harboring Tdc1 is reduced relative to wild type. Finally, we show that insertion of a transposable element is one of the mechanisms that can cause variation of plant cell cultures during repeated subculture. Received: 11 October 1996 / Accepted: 27 December 1996     

A novel cytoplasmic structure containing DNA networks in Entamoeba histolytica trophozoites

We report here the presence of cytoplasmic DNA arranged in networks in the trophozoites of the human parasite Entamoeba histolytica. Cytoplasmic DNA was detected in live trophozoites in a structure that we called EhkO, using the fluorescent dye acridine orange, and by in situ hybridization to trophozoites with a rDNA probe. The EhkO was found in the axenically grown clones A, L6 (strain HM1:IMSS) and MAVax (strain MAV) and in the polyxenically grown clone MAVpx (strain MAV). Bacteria present in MAVpx did not cross hybridize with the DNA probe neither in in situ hybridization or in Southern blot experiments. Autoradiography of metabolically [³H]thymidine-labeled trophozoites showed the presence of EhkO, and an EhkO-enriched fraction, purified from a nuclei-free extract and examined by light microscopy, exhibited [³H]thymidine incorporation into this structure. DNA was purified from the EhkO and enriched nuclear fractions and analyzed by transmission electron microscopy. The EhkO fraction contained DNA networks resembling those of trypanosome kDNA, whereas nuclear DNA was present mainly as linear molecules and some circles. Our findings imply that E. histolytica may be taxonomically more closely related to the Trypanosomatidae than previously suspected. Received: 7 August 1996 / Accepted: 7 October 1996     

Non-consumptive effects of larval Salamandra on crustacean prey: can eggs detect predators?

Predators affect prey populations not only by prey consumption but also in nonconsumptive ways including modifying prey behavior. I tested the effects of fire salamander larvae (Salamandra infraimmaculata) on populations of co-occurring crustacean species in artificial outdoor pools. I also tested whether these effects were due entirely to prey consumption by Salamandra larvae or alternatively to some nonconsumptive effect. The soil (containing crustacean eggs) added to the artificial pools was collected from a dried-out temporary pool that is inhabited by Salamandra during the early part of the hydroperiod. I randomly assigned the pools to one of three treatments: control, free Salamandra, or caged Salamandra. Free salamander larvae could roam the entire pool and prey upon crustaceans. Caged salamander larvae were placed within a cage with having 250-μm mesh windows. They could not prey upon the crustaceans but could, for example, influence them by chemical cues. Densities of the three dominant crustacean species (Arctodiaptomus similis, Ceriodaphnia quadrangula and Cyzicus sp.) were drastically reduced in both salamander treatments compared to the control. Crustacean densities, however, were not significantly different between the two salamander treatments. One plausible explanation is that crustacean eggs can detect the presence of this predator via chemical cues and delay hatching. Received: 4 March 1996 / Accepted, 23 October 1996     

A novel yeast gene, RHK1 , is involved in the synthesis of the cell wall receptor for the HM-1 killer toxin that inhibits β-1,3-glucan synthesis

The HM-1 killer toxin from Hansenula mrakii is known to inhibit cell wall β-1,3-glucan synthase of Saccharomyces cerevisiae and other sensitive strains of yeast. A number of mutants of Saccharomyces cerevisiae that show resistance to this toxin were isolated in order to clarify the killing mechanism of the toxin. These mutants, designated rhk (resistant to Hansenula killer), were classified into three complementation groups. A novel gene RHK1, which complements the killer-resistant phenotype of the largest complementation group rhk1, was isolated. DNA sequence analysis revealed an open reading frame that encodes a hydrophobic protein composed of 458 amino acids. Gene disruption followed by tetrad analysis showed that RHK1 is not essential and loss of RHK1 function endowed S. cerevisiae cells with complete killer resistance. A biochemical analysis suggested that RHK1 does not participate directly in the synthesis of β-1,3-glucan but is involved in the synthesis of the receptor for the HM-1 killer toxin. Received: 27 June 1996 / Accepted: 14 October 1996     

Role of dispersal ability in the phenotypic differentiation and plasticity of two marine gastropods

Species with contrasting abilities for dispersal may adopt different strategies in response to wide ranges of environmental conditions. These strategies were investigated here by comparisons of phenotypic differentiation and plasticity in the gastropods Austrocochlea constricta and Bembicium vittatum, which coexist in a range of intertidal habitats on the Abrolhos Islands and Albany in Western Australia. They differ in their potential for larval dispersal, A. constricta having a short mobile planktonic stage and B. vittatum developing directly from benthic eggs. Levels of among-population variation in shell shape and maximum size did not differ between the species, or between locations subject to contrasting levels of gene flow in the case of A. constricta. Results of a common garden translocation experiment, coupled with those of previous breeding experiments, suggest that variation in shape is predominantly genetically determined in B. vittatum, but plastic in A. constricta. Plasticity of shape in A. constricta was most pronounced at Albany, where previous data suggested the widest gene flow, and was greater than in B. vittatum at both locations. Change of shape in A. constricta subsequent to translocation was related to growth rate, a trait which was also more plastic in this species than in B. vittatum. The results agree with the notion that wide dispersal enhances physiological flexibility, while restricted dispersal promotes local genetic adaptation, but disagree with the hypothesis that wide dispersal promotes phenotypic homogeneity. Received: 31 May 1996 / Accepted: 30 September 1996     

Post-dispersal predation on Pinus sylvestris seeds by Fringilla spp: ground substrate affects selection for seed color

Background matching might lower the risk of seeds being eaten by seed predators that search visually. In aviary experiments, we analyzed the selection of diff erent-colored seeds by ground-feeding finches (Fringillacoelebs and F.montifringilla) against four naturally occurring forest soil substrates. The substrates were fresh burn (black), 6-year-old burn (brown), mineral soil (pale yellow) and Pleuroziumschreberi feather moss (green). We used color-sorted seeds of Pinussylvestris, a species with a large natural variation in seed color, ranging from pale yellow to black. Although seeds were scattered on the substrates at a density of only 91 seeds m⁻², birds removed seeds effectively. Both bird species found more pale than dark seeds on the fresh burn substrate. F. montifringilla also recovered more pale than dark seeds on the old burn, and more dark than pale seeds on mineral soil. In moss, the birds found very few seeds compared to the other substrates, and there was no color discrimination. P.sylvestris is frequently regenerating after fire, suggesting that dark seeds would be favored under selection from visually searching predators. Fire-adapted conifers with serotinous cones, e.g., Pinuscontorta ssp. latifolia, which spread their seeds primarily on freshly burnt surfaces, produce uniformly black or dark brown seeds. However, regeneration of the non-serotinous P.sylvestris is often extended for several years after a fire, during which substrate color and structure change. This may have helped to maintain variation in seed color. When regeneration of a plant species occurs on a substrate of uniform color, we believe that selection by visually searching seed predators will result in the evolution of cryptic seed color. Received: 16 August 1996 / Accepted: 30 November 1996     

Identification and genetic characterization of the Pseudomonas aeruginosaleuB gene encoding 3-isopropylmalate dehydrogenase

The Pseudomonas aeruginosa leuB gene, encoding 3-isopropylmalate dehydrogenase, was identified upstream of asd, encoding aspartate-β-semialdehyde dehydrogenase. Genetic analysis indicated that leuB is identical to the previously mapped gene defined by the leu-10 allele. The chromosomal leuB locus was inactivated by gene replacement. The insertions had no adverse effect on expression of the downstream asd gene but resulted in leucine auxotrophy. The leuB gene encodes a protein containing 360 amino acids (with a molecular weight of 39153), which was expressed in Escherichia coli as a M, 42000 protein. The results suggested that, in contrast to the situation in other bacteria (E. coli, Salmonella typhimurium and Bacillus subtilis) the P. aeruginosa leuB gene is physically separated from the genes encoding the other enzymes of the isopropylmalate pathway. Received: 15 August 1996 / Accepted: 23 October 1996     

Genetic analysis of sterile mutants in the dpy-5 unc-13 (I) genomic region of Caenorhabditis elegans

Essential genes were identified in the 1.5-map unit dpy-5 unc-13 region of chromosome I in the Caenorhabditis elegans genome by rescuing lethal mutations using the duplication sDp2. In this paper, we report the mapping and complementation testing of lethal mutations, 45 of which identify 18 new, essential genes. This analysis brings the number of essential genes defined by the sDp2 rescue of lethal mutants to 97; 64 of these map between dpy-5 and unc-13. 61% of these essential genes are identified by more than one allele. Positioning of the mutations was done using the breakpoints of six duplications. The mutant phenotypes of 14 loci essential for fertility were characterized by Nomarski microscopy and DAPI staining. None of the mutants were rescued by wild-type male sperm. The cytological data showed that four genes produced mutants with defects in gonadogenesis, let-395, let-603, let-605 and let-610. Mutations in seven genes, let-355, let-367, let-384, let-513, let-544, let-545 and let-606, affected germ cell proliferation or gametogenesis. Mutants for the remaining three genes, let-370, let-599 and let-604, produced eggs that failed to develop or hatch, thereby acting as maternal effect lethals. We observed a nonrandom distribution of arrest phenotypes with regard to map position. Received: 8 May 1996 / Accepted : 27 January 1997