A lethal allele at the putative regulatory locus, cpc-1, of cross-pathway control in Neurospora crassa

Summary A lethal allele at the putative regulatory locus, cpc-1, of cross-pathway control in Neurospora crassa was discovered by genetic analysis. cpc-1 ^j-5 is viable only in the presence of a second mutation, slo, causing slow growth. The detection of a lethal allele at a regulatory locus is a rare event and points to the physiological importance of the regulatory circuit concerned, namely the cross-pathway or general control of amino acid biosynthetic enzymes in lower eukaryotes.     

Somatic chimerism, genetic inheritance, and mapping of the fleshless berry (flb) mutation in grapevine (Vitis vinifera L.).

The fleshless berry (flb) mutation of grapevine (Vitis vinifera L. 'Ugni Blanc') impairs the differentiation and division of inner mesocarp cells responsible for flesh in grapevine berries. In order to study the inheritance of the mutation and to map the flb locus, 5 segregating populations were created. Progeny plants were classified as mutant or wild type by scoring for the presence of an ovary phenotype associated with the Flb- phenotype at anthesis. Phenotypic segregation revealed the involvement of a single dominant allele that was heterozygous in the original mutant. Through bulk segregant analysis, microsatellite (simple sequence repeat (SSR)) markers linked to the mutation were identified, and the flb locus was assigned to linkage group 18. The locus position was then refined by analyzing individual progeny and the segregation of SSR markers in the target region with the closest marker 5.6 cM distant from the flb locus. All progeny with the Flb- ovary phenotype differed from the original fleshless berry mutant in that no berries formed after anthesis. Our data suggest that the original mutant plant was a chimera with the mutated allele present in only 1 cell layer (L2 layer) of the ovary and berry.     

Genetic Analysis of Two Mutations Affecting Thymidine Metabolism in Dictyostelium Discoideum

The tmpA600 mutation confers thymidylate synthase deficiency and thymidine auxotrophy to Dictyostelium discoideum. The tdrA600 mutation enhances transport of thymidine and thereby reduces the auxotrophic requirement of tmpA600 strains. The tmpA locus maps to linkage group III. The tdrA600 mutation is dominant and cosegregates with both linkage groups IV and VI, possibly because of a translocation between the two. The tdrA600 allele is sufficient to allow efficient incorporation of exogenous [3H]thymidine or [3H]uridine into TCA-precipitable material and to sensitize the cell to the nucleoside-analog inhibitor, 5-fluorodeoxyuridine. These properties make the tdrA mutation useful for studies requiring labelling of DNA or RNA in vivo.     

Super Suppressors in NEUROSPORA CRASSA I. Induction, Genetic Localization and Relationship to a Missense Suppressor

Genetic analyses have been made to test the feasibility of using coincident reversions to prototrophy of multiple mutants to select super suppressors (ssu) in Neurospora crassa. Of five double-mutant strains examined, only those mutant combinations in which both members had the properties of nonsense mutations did revert coincidently. Forty-eight genetically purified coincident revertants were crossed to the wild type, and each was shown to contain a suppressor mutation. Five super suppressors were examined more thoroughly. Tetrad and random spore analysis was used to demonstrate that each behaved as a single gene in crosses. Two super suppressors, ssu-1 and ssu-4 were localized respectively on the right and left arm of linkage group 7. Two others, ssu-2 and ssu-3, appear to map on the right arm of linkage group 1. The fifth super suppressor mapped, ssu-7, lies between ad-8 and ylo-1 on linkage group 6. One super suppressor, ssu-1, was interesting because it mapped near the location reported for the suppressor of the missense mutant tryp-3(td201) (Yourno and Suskind 1964a). However, no overlap was found in action spectrum of the two suppressors. Tetrad analysis showed the two suppressors were located about 10 map units apart, the missense suppressor being the more distal to the centromere.     

Genetic analysis and molecular mapping of a pale flower allele at the W4 locus in soybean.

In soybean (Glycine max (L.) Merr.), the w4-mutable line that harbors the w4-m allele was identified in 1983. It was proposed that this line contained an autonomous transposable element at the W4 locus, which is a major locus controlling the biosynthesis of anthocyanin. The w4-m allele can revert to the W4 allele that produces the wild-type phenotype, or sometimes to other alleles that produce intermediate phenotypes. Mutant plants that produce pale flowers were identified among the progeny of a single germinal revertant event from the w4-mutable line. Through genetic analysis, we established that the pale-flower mutation was conditioned by a new allele (w4-p) at the W4 locus. The w4-p allele is dominant to the w4 allele but recessive to the W4 allele, and the w1 allele has an epistatic effect on the w4-p allele. The pale-mutant line (w4-pw4-p) was designated as Genetic Type Collection number T369. An F2 mapping population derived from the cross of Minsoy (W4W4) x T369 (w4-pw4-p) was used to map the W4/w4-p locus, using simple sequence repeat (SSR) markers. The W4 locus was located at one end of molecular linkage group D2, 2.3 cM from the SSR marker Satt386 and close to the nearby telomere.     

The targeted disruption of the CD98 gene results in embryonic lethality

CD98 is one of the important molecules for development, cell differentiation, cell proliferation, and regulation of cellular function. In this study, CD98 heavy chain (HC) knockout mice were produced and analyzed. Five targeted ES clones were obtained and colony frequency was about 2%. One (clone 113) of the five heterozygous ES cell clones had undergone aberrant recombination at the 5' side. The aberrant recombination happened at the site between second intron and 5' arm. All lines from correctly targeted clones could not transmit the mutated allele to spermatozoa. The mutated allele derived from the aberrant targeted clone was transmitted to the progeny. However, none of the F2 mice was homozygous for the CD98 mutation, indicating that the targeted disruption of the CD98 gene results in embryonic lethality. The point of embryonic lethality is considered to be between 3.5 and 9.5 dps. These findings indicate that CD98 molecules are essential for mouse embryogenesis.     

Extragenic suppressors of paralyzed flagellar mutations in Chlamydomonas reinhardtii identify loci that alter the inner dynein arms

We have analyzed extragenic suppressors of paralyzed flagella mutations in Chlamydomonas reinhardtii in an effort to identify new dynein mutations. A temperature-sensitive allele of the PF16 locus was mutagenized and then screened for revertants that could swim at the restrictive temperature (Dutcher et al. 1984. J. Cell Biol. 98:229-236). In backcrosses of one of the revertant strains to wild-type, we recovered both the original pf16 mutation and a second, unlinked suppressor mutation with its own flagellar phenotype. This mutation has been identified by both recombination and complementation tests as a new allele of the previously uncharacterized PF9 locus on linkage group XII/XIII. SDS-PAGE analysis of isolated flagellar axonemes and dynein extracts has demonstrated that the pf9 strains are missing four polypeptides that form the I1 inner arm dynein subunit. The primary effect of the loss of the I1 subunit is a decrease in the forward swimming velocity due to a change in the flagellar waveform. Both the flagellar beat frequency and the axonemal ATPase activity are nearly wild-type. Examination of axonemes by thin section electron microscopy and image averaging methods reveals that a specific domain of the inner arm complex is missing in the pf9 mutant strains (see accompanying paper by Mastronarde et al.). When combined with other flagellar defects, the loss of the I1 subunit has synergistic effects on both flagellar assembly and flagellar motility. These synthetic phenotypes provide a screen for new suppressor mutations in other loci. Using this approach, we have identified the first interactive suppressors of a dynein arm mutation and an unusual bypass suppressor mutation.     

Likelihood analysis of disequilibrium mapping, and related problems.

In this paper a theory is developed that provides the sampling distribution of alleles at a diallelic marker locus closely linked to a low-frequency allele that arose as a single mutant. The sampling distribution provides a basis for maximum-likelihood estimation of either the recombination rate, the mutation rate, or the age of the allele, provided that the two other parameters are known. This theory is applied to (1) the data of Hästbacka et al., to estimate the recombination rate between a locus associated with diastrophic dysplasia and a linked RFLP marker; (2) the data of Risch et al., to estimate the age of a presumptive allele causing idiopathic distortion dystonia in Ashkenazi jews; and (3) the data of Tishkoff et al., to estimate the date at which, at the CD4 locus, non-African lineages diverged from African lineages. We conclude that the extent of linkage disequilibrium can lead to relatively accurate estimates of recombination and mutation rates and that those estimates are not very sensitive to parameters, such as the population age, whose values are not known with certainty. In contrast, we also conclude that, in many cases, linkage disequilibrium may not lead to useful estimates of allele age, because of the relatively large degree of uncertainly in those estimates.     

A perithecial color mutant of Neurospora crassa

Summary A mutation, per-1, was found in N. crassa which caused orange instead of black perithecia when the protoperithecial parent contained the per-1 allele. No effect of per-1 was found on vegetative morphology or nutritional requirements. The per-1 locus was mapped proximal to iv (?6201) in the right arm of linkage group V.     

Recurrent locus-specific mutation resulting from a cryptic ectopic insertion in Neurospora

New mutations are found among approximately 20% of progeny when one or both parents carry eas allele UCLA191 (eas(UCLA), easily wettable, hydrophobin-deficient, linkage group II). The mutations inactivate the wild-type allele of cya-8 (cytochrome aa3 deficient, linkage group VII), resulting in thin, "transparent" mycelial growth. Other eas alleles fail to produce cya-8 mutant progeny. The recurrent cya-8 mutations are attributed to repeat-induced point mutation (RIP) resulting from a duplicated copy of cya-8+ that was inserted ectopically at eas when the UCLA191 mutation occurred. As expected for RIP, eas(UCLA)-induced cya-8 mutations occur during nuclear proliferation prior to karyogamy. When only one parent is eas(UCLA), the new mutations arise exclusively in eas(UCLA) nuclei. Mutation of cya-8 is suppressed when a long unlinked duplication is present. Stable cya-8 mutations are effectively eliminated in crosses homozygous for rid, a recessive suppressor of RIP. The eas(UCLA) allele is associated with a long paracentric inversion. A discontinuity is present in eas(UCLA) DNA. The eas promoter is methylated in cya-8 progeny of eas(UCLA), presumably by the spreading of methylation beyond the adjoining RIP-inactivated duplication. These findings support a model in which an ectopic insertion that created a mutation at the target site acts as a locus-specific mutator via RIP.     

Genetic mapping a meiotic driver that causes sex ratio distortion in the mosquito Aedes aegypti

An endogenous meiotic driver in the dengue and yellow fever vector mosquito Aedes aegypti can cause highly male-biased sex ratio distortion in crosses from suitable genetic backgrounds. We previously selected a strain that carries a strong meiotic drive gene (D) linked with the male-determining allele (M) on chromosome 1 in A. aegypti. Here, we performed segregation analysis of the M(D) locus among backcross (BC(1)) progeny from a driver male and drive-sensitive females. Assessment of sex ratios among BC(2) progeny showed ～5.2% recombination between the M(D) locus and the sex determination locus. Multipoint linkage mapping across this region revealed consistent marker orders and recombination frequencies with the existing reference linkage map and placed the M(D) locus within a 6.5-cm interval defined by the LF159 locus and microsatellite marker 446GAA, which should facilitate future positional cloning efforts.     

Microsatellite mapping of the powdery mildew resistance gene <Emphasis Type="Italic">Pm5e</Emphasis> in common wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Powdery mildew, caused by Erysiphe graminis DM f. sp. tritici (Em. Marchal), is one of the most important diseases of common wheat world-wide. Chinese wheat variety 'Fuzhuang 30' carries the powdery mildew resistance gene Pm5e and has proven to be a valuable resistance source of powdery mildew for wheat breeding. Microsatellite markers were employed to identify the gene Pm5e in a F(2) progeny from the cross 'Nongda 15' (susceptible) x 'Fuzhuang 30' (resistant). The gene Pm5e was mapped in the distal region of chromosome 7BL. Seven microsatellite markers were found to be linked to the gene Pm5e, of which two codominant markers Xgwm783 and Xgwm1267 were relatively close to Pm5e with a linkage distance of 11.0 cM and 6.6 cM, respectively. It is possible to use the 136-bp allele of Xgwm1267 in 'Fuzhuang 30' for marker-assisted selection during the wheat resistance breeding process for facilitation of gene pyramiding. The mapping information in the present study provides a starting point for fine mapping of the Pm5 locus and map-based cloning to clarify the molecular structure and function of the different alleles at the Pm5 locus. A microsatellite linkage map of chromosome 7B was constructed with 20 microsatellite loci, nine on the short arm and 11 on the long arm. This information will be very useful for further mapping of agronomically important genes of interest on chromosome 7B.     

Genetics of rye phosphatases: evidence of a duplication

Summary Genetic analyses were conducted on alkaline phosphatases of the endosperm of dry kernels and leaf acid phosphatases in four open pollinated and one inbred line of cultivated rye (Secale cereale L.). A total of seven alkaline phosphatase isozymes were observed occurring at variable frequencies in the different cultivars analyzed. We propose that at least five loci control the alkaline phosphatases of rye endosperm — Alph-1, Alph-2, Alph-3, Alph-4 and Alph-5 — all of which have monomeric behaviour. The leaf acid phosphatases are controlled by one locus and have a dimeric quaternary structure. All loci coding for alkaline phosphatase isozymes showed one active, dominant allele and one null, recessive allele, except for the locus Alph-3 which showed two active, dominant alleles and one null, recessive one. The linkage analyses suggest the existence of two linkage groups for alkaline phosphatases: one of them would contain Alph-2, Alph-4, Alph-5 and the locus/loci coding isozymes 6 and 7. This linkage group is located in the 7RS chromosome arm. The other group would include Alph-1 and Alph-3 loci, being located in the 1RL chromosome arm. Leaf acid phosphatases have been previously located in the 7RL chromosome arm. Our data also support an independent relationship between loci controlling the endosperm alkaline phosphatases and leaf acid phosphatases.     

Changes in gene expression elicited by amino acid limitation in Neurospora crassa strains having normal or mutant cross-pathway amino acid control

Summary The effects of amino acid limitation on gene expression have been investigated in Neurospora crassa strains carrying normal (cpc-1 ⁺) or mutant (cpc-1) alleles at a locus implicated in cross-pathway amino acid control. Electrophoresis and fluorography were used to reveal the patterns of label incorporation into polypeptides in vivo, or after in vitro translation of extracted mRNAs. In a cpc-1 ⁺ strain at least 20% of detectable in vitro translation products showed relative increases in incorporation when RNA was obtained from mycelium grown under conditions of arginine limitation, by comparison with conditions of arginine sufficiency. A cpc-1 mutation, which impairs derepression of a variety of amino acid synthetic enzymes following amino acid limitation, had little detectable effect on in vivo polypeptide synthesis during amino acid sufficient growth or following pyrimidine limitation. However the mutation substantially altered the response to arginine or histidine limitation. The majority of in vitro translation products that showed increased expression in arginine limited cpc-1 ⁺ failed to increase in cpc-1 strains, but arginine limitation of cpc-1 also resulted in increases that did not occur in cpc-1 ⁺ strains. This may reflect both direct and indirect consequences of the impairment of cross-pathway control.     

Synteny on mouse chromosome 5 of homologs for human DNA loci linked to the Huntington disease gene

Comparative mapping in man and mouse has revealed frequent conservation of chromosomal segments, offering a potential approach to human disease genes via their murine homologs. Using DNA markers near the Huntington disease gene on the short arm of chromosome 4, we defined a conserved linkage group on mouse chromosome 5. Linkage analyses using recombinant inbred strains, a standard outcross, and an interspecific backcross were used to assign homologs for five human loci, D4S43, D4S62, QDPR, D4S76, and D4S80, to chromosome 5 and to determine their relationships with previously mapped markers for this autosome. The relative order of the conserved loci was preserved in a linkage group that spanned 13% recombination in the interspecific backcross analysis. The most proximal of the conserved markers on the mouse map, D4S43h, showed no recombination with Emv-1, an endogenous ecotropic virus, in 84 outcross progeny and 19 recombinant inbred strains. Hx, a dominant mutation that causes deformities in limb development, maps approximately 2 cM proximal to Emv-1. Since the human D4S43 locus is less than 1 cM proximal to HD near the telomere of chromosome 4, the murine counterpart of the HD gene might lie between Hx and Emv-1 or D4S43h. Cloning of the region between these markers could generate new probes for conserved human sequences in the vicinity of the HD gene or possibly candidates for the murine counterpart of this human disease locus.     

Mutations of MYO6 are associated with recessive deafness,DFNB37

Cosegregation of profound, congenital deafness with markers on chromosome 6q13 in three Pakistani families defines a new recessive deafness locus, DFNB37. Haplotype analyses reveal a 6-cM linkage region, flanked by markers D6S1282 and D6S1031, that includes the gene encoding unconventional myosin VI. In families with recessively inherited deafness, DFNB37, our sequence analyses of MYO6 reveal a frameshift mutation (36-37insT), a nonsense mutation (R1166X), and a missense mutation (E216V). These mutations, along with a previously published missense allele linked to autosomal dominant progressive hearing loss (DFNA22), provide an allelic spectrum that probes the relationship between myosin VI dysfunction and the resulting phenotype.