Probe,VK5B,is located in the same interval as the autosomal dominant adult polycystic kidney disease locus,PKD1

Summary The polymorphic DNA probe VK5B (D16S94) was mapped by genetic linkage in families from the Centre d'Etude de Polymorphisme Humain (CEPH) as being in the same interval as the autosomal dominant adult polycystic kidney disease locus (PKD1). The maximum likelihood estimate of the genetic location of VK5B using multipoint linkage analysis was 9.6cM proximal to {ie286-01} (D16S85) and 5.4cM distal to CRI-0327 (D16S63), in males. The VK5B probe may be useful in PKD1 families for prenatal and presymptomatic diagnosis of the disease. Additional typing of PKD1 families is required to determine whether the location of VK5B is distal or proximal to (PKD1).     

Differential adaptation to water deprivation in first-instar nymphs of the German cockroach (Blattella germanica) and the brown-banded cockroach (Supella longipalpa)

This study compares the relative abilities of rist-instar nymphs of the german cockroach (Blattella germanica L.) and the brown-banded cockroach (Supella longipalpa F.) to survive prolonged periods of food and/or water deprivation.S. longipalpa nymphs survived significantly longer thanB. germanica nymphs when drinking water was absent, but the advantage ofS. longipalpa overB. germanica disappeared if food was also absent. These results are consistent with the hypothesis thatS. longipalpa is adapted to drier habitats than isB. germanica, and suggest thatS. longipalpa nymphs may be more capable of producing and utilizing extra metabolic water from food thanB. germanica nymphs. Evidence from the literature suggests that this species difference applies mainly to juveniles, since adults ofB. germanica andS. longipalpa do not differ in ability to withstand water deprivation, regardless of food availability.     

Inheritance of enzymes and blood proteins in the leopard frog,Rana pipiens: Three linkage groups established

Individuals from natural populations of the leopard frog, Rana pipiens, were analyzed for electrophoretic differences in blood proteins and enzymes from an amputated digit. The proteins examined represent products of 72 loci. Presumptive heterozygotes at multiple loci were selected for experimental crosses. Mendelian inheritance of 18 protein variations were demonstrated in the offspring. Tests for linkage or independent assortment were performed for 75 locus pairs. Three linkage groups were established. Linkage group 1 contains two loci, aconitase-1 (Acon1) and serum albumin (Alb), with a 19% recombination frequency between them. Linkage group 2 contains four loci, glyoxalase (Gly), acid phosphatase-1 (Ap1), acid phosphatase-2 (AP2), and esterase-5 (Est5). The data show the relationships Gly-21.1%-AP1-0%-AP2-6.3%-Est5, and Gly-25.6%-Est5. Linkage group 3 consists of four closely linked esterase loci. The data, Est1-5.1%-Est6, Est6-1.8%-Est10-1.9%-Est4 and Est6-3.0%-Est4, do not establish a complete order but suggest that Est10 is between Est4 and Est6. These results, with data demonstrating apparent independent assortment of 67 other locus pairs, provide a foundation for establishing the frog genetic map.The project was supported by Grant No. RR-00572 from the Division of Research Resources, National Institutes of Health. This paper is contribution No. C-87 from the Amphibian Facility, George W. Nace, Director.     

Linkage analyses using biochemical variants in mice. III. Linkage relationships of eleven biochemical markers

The linkage relationships of 11 loci concerned with protein or enzyme variation in the inbred mouse (Mus musculus) have been investigated. By means of a three-point cross, the order of the loci glucosephosphate isomerase (Gpi-1), albino (c), and hemoglobin -chain in linkage group I has been established as Gpi-c-Hbb. Similarly, the order of the loci autosomal glucose 6-phosphate dehydrogenase (Gpd-1), misty (m), and brown (b) in linkage group VIII has been established as Gpd-m-b. The levulinate dehydratase locus (Lv) in linkage group VIII which shows 5±2% recombination with the brown locus is near the anemia locus (an). The locus for malic dehydrogenase (Mdh-1) shows 10.1±2.9% recombination with the dilute locus and 12.0±6.5% recombination with the luxoid locus. The tentative order of the three loci is d-Mdh-1-lu. Recombination between the isocitric dehydrogenase locus (Id-1) and the leaden locus (ln) is 16.7±5.8% and between Id-1 and the splotch locus (Sp) is 11.0±5.4% in linkage group XIII. The tentative order of the three loci is ln-Sp-Id-1. Recombination between the lactic dehydrogenase regulatory locus (Ldr-1) and the microphthalmia locus (Mi ^wh) in linkage group XI is 28.7±4.4%. Recombination between the phosphoglucomutase locus (Pgm-1) and the W-locus in linkage group XVII is 3.0±1.7%. The esterase-3 locus has not been placed in a linkage group and has been tested against markers on linkage groups I, II, III, IV, V, VI, VIII, XI, XII, XIII, XVI, XVII, XVIII, and XX. In no case was there physical linkage of structural genes whose products participate in related metabolic pathways.Supported by the Roche Institute of Molecular Biology and AEC contract AT (30-1)-3671 with The Jackson Laboratory. The principles of laboratory animal care as promulgated by the National Society for Medical Research were observed.To Dr. Margaret M. Dickie—in memoriam.     

Linkage analysis of the myosin heavy chain gene in Dictyostelium discoideum using a mutation generated by homologous recombination

Summary A mutation (mhcA1 in strain HMM) created by insertional gene inactivation was used to map the Dictyostelium discoideum myosin heavy chain gene (mhcA) to linkage group IV. Three phenotypic traits associated with this mutation (slow colony growth, inability of the mutant to develop past aggregation, and the presence of five to ten integrated vector copies) cosegregated as expected for the consequences of a single insertional event. This linkage was confirmed using a restriction fragment length polymorphism. The mhcA1 mutation was recessive to wild type and was nonallelic with mutations at the following loci on linkage group IV: aggJ, aggL, couH, minA, phgB and tsgB. This work demonstrates the ability to apply standard techniques developed for D. discoideum parasexual genetic analyses to mutants generated by transformation, which is of particular relevance to analysis of genes for which no classical mutations or restriction fragment length polymorphisms are available.     

Mutations in tumour suppressor genes,l(2)gl andl(2)gd,alter the expression ofwingless inDrosophila

To understand the roles of two well known tumour suppressor genes.l(2)gl andl(2)gd in normal imaginal disc development inDrosophila, we have initiated a study to examine effect of mulations of these genes on the expression of genes involved in the patterning of the imaginal discs. In this study we show that the expression ofwingless, theDrosophila orthologue of the mammalian oncogeneWnt, is affected in the imaginal discs ofl(2)gl ⁴ andl(2)gd ¹ mutant individuals. In the tumourous wing imaginal discs froml(2)gl mutant larvae, the pattern ofwingless expression was progressively disrupted with an increase in the area of expression, Tumourous wing imaginal discs froml(2)gd homozygous individuals exhibited progressive broadening and extension of the wingless expressing domains. We suggest thatl(2)gl andl(2)gd might be involved in regulating post embryonic expression ofWingless.     

Interactions of thePolycomb group of genes with homeotic loci ofDrosophila

Summary Members of thePolycomb (Pc) group of genes are required for the correct determination of segment identity, and are thought to be negative regulators of thebithorax andAntennapedia complexes. This hypothesis has been tested molecularly for only some members of thePc group. Here, we examine the distribution ofUltrabithorax (Ubx),Antennapedia (Antp), andSex combs reduced (Scr) proteins in the epidermis, central nervous system, and midgut of embryos homozygous for mutations in tenPc group genes. We show that zygotic loss of mostPc group genes causes ectopic expression ofUbx andAntp, but that there are differences in time and tissue-specificity. FivePc group mutations lack midgut constrictions and also exhibit ectopic or suppressedUbx expression and suppression ofAntp expression. Distribution ofAntp is upset earlier than distribution ofUbx in the central nervous system of everyPc group mutant affecting both genes. Loss of the zygotic products ofPolycomb, extra sex combs, andAdditional sex combs cause ectopic expression ofScr in epidermis, and allPc group genes exceptPsc have suppressedScr expression in the nervous system. These results are discussed with respect to the function of thePc group.     

Mutations in somePolycomb group genes ofDrosophila interfere with regulation of segmentation genes

Mutations in severalPolycomb (Pc) group genes cause maternal-effect or zygotic segmentation defects, suggesting thatPc group genes may regulate the segmentation genes ofDrosophila. We show that individuals doubly heterozygous for mutations inpolyhomeotic and six otherPc group genes show gap, pair rule, and segment polarity segmentation defects. We examined double heterozygous combinations ofPc group and segmentation mutations for enhancement of adult and embryonic segmentation defects.Posterior sex combs andpolyhomeotic interact withKrüppel ² and enhance embryonic phenotypes ofhunchback andknirps, andpolyhomeotic enhanceseven-skipped. Surprisingly, flies carrying duplications ofextra sex combs (esc), that were heterozygous for mutations ofeven-skipped (eve), were extremely subvital. Embryos and surviving adults of this genotype showed strong segmentation defects in even-numbered segments. Antibody studies confirm that expression ofeve is suppressed by duplications ofesc. However,esc duplications have no effect on other gap or pair rule genes tested. To our knowledge, this is only the second triplo-abnormal phenotype associated withPc group genes. Duplications of nine otherPc group genes have no detectable effect oneve. Expression ofengrailed (en) was abnormal in the central nervous systems of mostPc group mutants. These results support a role forPc genes in regulation of some segmentation genes, and suggest thatesc may act differently from otherPc group genes.     

A full saturated linkage map of<Emphasis Type="Italic"> Picea abies</Emphasis> including AFLP,SSR, ESTP, 5S rDNA and morphological markers

Based on an F₁ progeny of 73 individuals, two parental maps were constructed according to the double pseudo-test cross strategy. The paternal map contained 16 linkage groups for a total genetic length of 1,792 cM. The maternal map covered 1,920 cM, and consisted of 12 linkage groups. These parental maps were then integrated using 66 intercross markers. The resulting consensus map covered 2,035 cM and included 755 markers (661 AFLPs, 74 SSRs, 18 ESTPs, the 5S rDNA and the early cone formation trait) on 12 linkage groups, reflecting the haploid number of chromosomes of Picea abies. The average spacing between two adjacent markers was 2.6 cM. The presence of 39 of the SSR and/or ESTP markers from this consensus map on other published maps of different Picea and Pinus species allowed us to establish partial linkage group homologies across three P. abies maps (up to five common markers per linkage group). This first saturated linkage map of P. abies could be therefore used as a support for developing comparative genome mapping in conifers.Communicated by O. Savolainen     

Characterization of type II protein secretion (xcp) genes in the plant growth-stimulatingPseudomonas putida, strain WCS358

InPseudomonas aeruginosa, the products of thexcp genes are required for the secretion of exoproteins across the outer membrane. Despite structural conservation of the Xcp components, secretion of exoproteins via the Xcp pathway is generally not found in heterologous organisms. To study the specificity of this protein secretion pathway, thexcp genes of another fluorescent pseudomonad, the plant growth-promotingPseudomonas putida strain WCS358, were cloned and characterized. Nucleotide sequence analysis revealed the presence of at least five genes, i.e.,xcpP, Q, R, S, andT, with homology toxcp genes ofP. aeruginosa. Unlike the genetic organization inP. aeruginosa, where thexcp cluster consists of two divergently transcribed operons, thexcp genes inP. putida are all oriented in the same direction, and probably comprise a single operon. Upstream ofxcpP inP. putida, an additional open reading frame, with no homolog inP. aeruginosa, was identified, which possibly encodes a lipoprotein. Mutational inactivation ofxcp genes inP. putida did not affect secretion, indicating that no proteins are secreted via the Xcp system under the growth conditions tested, and that an alternative secretion system is operative. To obtain some insight into the secretory pathway involved, the amino acid sequence of the N-terminus of the major extracellular protein was determined. The protein could be identified as flagellin. Mutations in thexcpQ andR genes ofP. aeruginosa could not be complemented by introduction of the correspondingxcp genes ofP. putida. However, expression of a hybrid XcpR protein, composed of the N-terminal one-third ofP. aeruginosa XcpR and the C-terminal two-thirds ofP. putida XcpR, did restore protein secretion in aP. aeruginosa xcpR mutant.     

A functional<Emphasis Type="Italic"> gltB</Emphasis> gene is essential for utilization of acidic amino acids and expression of periplasmic glutaminase/asparaginase (PGA) by<Emphasis Type="Italic"> Pseudomonas putida</Emphasis> KT2440

Pseudomonas putida KT2440, a root-colonizing fluorescent pseudomonad, is capable of utilizing acidic amino acids (Asp and Glu) and their amides (Asn and Gln) as its sole source of carbon and nitrogen. The uptake of Gln and Asn is facilitated by a periplasmic glutaminase/asparaginase (PGA), which hydrolyses Asn and Gln to the respective dicarboxylates. Here, we describe transposon mutagenesis of P. putida KT2440 with a self-cloning promoter probe vector, Tn5-OT182. Transconjugants defective in Glu-mediated PGA induction were selected for further studies. In most clones the transposon was found to have integrated into the gltB gene, which encodes the major subunit of the glutamate synthase (GOGAT). The transconjugants were nonmotile, no longer showed a chemotactic response towards amino acids, and could not survive prolonged periods of starvation. The acidic amino acids and their amides supported growth of the transconjugants only when supplied together with glucose, suggesting that the gltB-mutants had lost the ability to utilize amino acids as a carbon source. To confirm that gltB inactivation was the cause of this phenotype, we constructed a mutant with a targeted disruption of gltB. This strain behaved like the clones obtained by random mutagenesis, and failed to express not only PGA but also a number of other Glu-induced proteins. In contrast to wild-type cells, the gltB ^- strain accumulated considerable amounts of both Glu and Gln during long-term incubation.Communicated by A. Kondorosi     

Expression of parasporal crystal protein (δ—endotoxin) gene(s) ofBacillus thuringiensis var.israelensis in sporogenic and asporogenic mutant strains ofBacillus cereus

The expression of parasporal crystal protein (δ-endotoxin) coding gene(s) ofBacillus thuringlensis var.israelensis and its association, if any, with sporulation was studied in sporogenicBacillus cereus and its asporogenic mutant strains. Five asporogenous mutants ofBacillus cereus blocked at different stages of sporulation, were isolated from a streptomycin-resistant strain, The transconjugants isolated from the plasmid transfer experiments betweenBacillus thuringiensis var.israelensis and streptomycin resistantBacillus cereus and its asporogenous mutants, showed larvicidal activity. The crystal protein gene(s) are, therefore, expressed both in sporulating and in non-sporulating mutant strains ofBacillus cereus suggesting that the expression of crystal protein gene(s), is independent of sporulation specific functions inBacillus cereus. Part of the work was carried out at Biotechnology Programme, Jadavpur University, Calcutta 700 032, India.     

Genetic analysis of Aspergillus niger: Isolation of chlorate resistance mutants, their use in mitotic mapping and evidence for an eighth linkage group

Summary This paper describes the use of chlorate resistant mutants in genetic analysis of Aspergillus niger. The isolated mutants could be divided into three phenotypic classes on the basis of nitrogen utilization. These were designated nia, nir and cnx as for Aspergillus nidulans. All mutations were recessive to their wild-type allele in heterokaryons as well as in heterozygous diploids. The mutations belong to nine different complementation groups. In addition a complex overlapping complementation group was found. Evidence for the existence of eight linkage groups was obtained. Two linked chlorate resistance mutations and two tryptophan auxotrophic markers, which were unlinked to any of the known markers (Goosen et al. 1989), form linkage group VIII. We used the chlorate resistance mutations as genetic markers for the improvement of the mitotic linkage map of A. niger. We determined the linear order of three markers in linkage group VI as well as the position of the centromere by means of direct selection of homozygous cnxA1 recombinants. In heterozygous diploid cultures diploid chlorate resistant segregants appeared among conidiospores with a frequency of 3.9×10^–2 (cnxG13 in linkage group I) to 2.1 × 10^–2 (cnxD6 in linkage group 111). The mean frequency of haploid chlorate resistant segregants was 1.3 × 10^–3. The niaD1 and niaD2 mutations were also complemented by transformation with the A. niger niaD ⁺ gene cloned by Unkles et al. (1989). Mitotic stability of ten Nia⁺ transformants was determined. Two distinct stability classes were found, showing revertant frequencies of 5.0 × 10^–3 and 2.0 × 10^–5 respectively.     

Efficient transformation of the medicinal mushroomGanoderma lucidum

Ganoderma lucidum is a well-known and important medicinal mushroom, but its genetic modification has not been reported. We developed an efficient procedure for isolation and regeneration of protoplasts fromG. lucidum. To construct a vector for high-level expression of heterologous genes inG. lucidum, the 1.4-kb regulatory region of the glyceraldehyde-3-phosphate dehydrogenase gene (GPD) was isolated from the genomic DNA ofLentinus edodes, and theGPD promoter was fused to the β-glucuronidase (GUS) and bialaphos resistance (bar) genes. Using the resulting construct, p301-bG1, an efficient transformation system based on electroporation was established forG. lucidum. GUS expression was observed among transformants conferring bialaphos resistance, indicating that theL. edodes GPD promoter can be used for expression of exogenous genes inG. lucidum. We also studied green fluorescent protein (GFP) as another reporter for transformation ofG. lucidum. TheL. edodes GPD promoter was fused respectively to theGFP andbar genes, and the resulting construct, p301-bg, was introduced intoG. lucidum. StableGFP expression in transformants was detectable by fluorescence microscopy, suggesting the suitability ofGFP as a reporter system in transformation of this mushroom. This is the first report of an efficient transformation system forG. lucidum using different reporters, paving the way for genetic modification of this famous medicinal mushroom.     

Foraging and spatial dispersion in protein-scavenging workers ofVespula germanica andV. maculifrons (Hymenoptera: Vespidae)

A series of experiments on the discovery of meat bait by scavenging workers ofV. germanica andV. maculifrons showed that workers oriented to meats by visual cues and olfactory cues. Scavening workers ofV. germanica andV. maculifrons responded positively to the sight of conspecifics at meat baits; i.e., meat with (confined) wasps received more visits than meat without wasps. This attraction to conspecific workers on meat baits provides evidence of local enhancement, in that the presence of conspecifics at meat bait directs other wasps to a particular spatial locality. Local enhancement by yellowjackets foraging for meat may contribute to the clumped distribution of worker populations noted in studies of workers captured in meat-baited traps. We hypothesize that local enhancement of foraging is a ubiquitous behavior in species of theV. vulgaris species group and may be one attribute explaining their ecological success.     

The isolation and analysis of one functional type ofpyr-3 mutant inNeurospora

Twenty-seven pyrimidine-requiring mutants were isolated as suppressors of anarg-3 mutant. All 27 are deficient for ATCase activity and show linkage to thecol-4 marker located on linkage group IV. Analyses of prototroph frequencies resulting from crosses of the new mutants to previously mappedpyr-3 mutants indicate that this functional type ofpyr-3 mutant is restricted to one region of the genetic map. Complementation studies with 11 of the new mutants further extend and subdivide the complementation map of thepyr-3 locus.This work was supported in part by National Institutes of Health, Public Health Service Grant GM 15137-01 and by National Science Foundation Grant GB5998.     

Distribution,inheritance and linkage relationships of ribosomal DNA spacer length variants in pea

Summary DNA restriction endonuclease fragment analysis is used to examine the genetic organization, inheritance and linkage associations of the ribosomal DNA in pea. The substantial variation observed in the length of the intergenic spacer region is shown to segregate in Mendelian fashion involving two independent genetic loci, designated Rrn1 and Rrn2. Linkage between Rrn1 and two marker loci on chromosome 4 establishes the approximate location of this tandem array. Rrn2 shows linkage with a set of isozyme loci which assort independently of other markers on all seven chromosomes. Combining these observations with previous cytological data, we suggest that Rrn2 and the isozyme loci linked to it constitute a new linkage group on chromosome 7. The general absence of spacer length classes common to both rRNA loci in any of the lines we examined indicates that little or no genetic exchange occurs between the nonhomologous nucleolar organizer regions.     

Nitrite toxicity inAspergillus nidulans: Effect of mutation at thenihB gene

ThenihB gene ofAspergillus nidulans was found to confer sensitivity to elevated concentrations of nitrite, compact morphology and absence of conidiation. ThenihB locus was allocated to linkage group II and was recessive in heterozygous diploids. When thenihB1 mutant was grown on a mixture of nitrite plus NH ₄ ⁺ its sensitivity to nitrite was unchanged. A possible role for this gene in nitrite transport and/or the maintenance of membrane integrity is discussed.