Complementation Analysis of Linked Circadian Clock Mutants of NEUROSPORA CRASSA

A fourth mutant of Neurospora crassa, designated frq-4, has been isolated in which the period length of the circadian conidiation rhythm is shortened to 19.3 ± 0.3 hours. This mutant is tightly linked to the three previously isolated frq mutants, and all four map to the right arm of linkage group VII about 10 map units from the centromere. Complementation tests suggest, but do not prove, that all four mutations are allelic, since each of the four mutants is co-dominant with the frq⁺ allele—i.e., heterokaryons have period lengths intermediate between the mutant and wild-type—and since heterokaryons between pairs of mutants also have period lengths intermediate between those of the two mutants.     

Chromosome Fragments in DICTYOSTELIUM DISCOIDEUM Obtained from Parasexual Crosses between Strains of Different Genetic Background

The first aneuploid strains of Dictyostelium discoideum have been unambiguously characterized, using cytological and genetic analysis. Three independently isolated, but genetically similar, fragment chromosomes have been observed in segregants from diploids formed between haploid strains derived from the NC4 and V12 isolates of D. discoideum. Once generated, the fragment chromosomes, all of which have V12-derived centromeres, can be maintained in a NC4 genetic background. Genetic evidence is consistent with the view that all three fragment chromosomes studied encompass the region from the centromere to the whiA locus of linkage group II and terminate in the interval between whiA and acrA. From cytological studies, one of the fragment chromosomes consists of approximately half of linkage group II.—We observed no deleterious effect on viability or asexual fruiting-body formation in either haploid or diploid strains carrying an additional incomplete chromosome and hence are disomic or trisomic, respectively, for part of linkage group II. The incomplete chromosome is lost at a frequency of 2 to 3% from disomic and trisomic strains, but surprisingly this loss is not increased in the presence of the haploidizing agent, benlate. A new locus (clyA), whose phenotype is altered colony morphology, is assigned to the region of linkage group II encompassed by the fragment chromosome.     

Linkage Group Xix of Chlamydomonas Reinhardtii Has a Linear Map

Linkage group XIX (or the UNI linkage group) of Chlamydomonas reinhardtii has been reported to show a circular meiotic recombination map. A circular map predicts the existence of strong chiasma and chromatid interference, which would lead to an excess number of two-strand double crossovers during meiosis. We have tested this prediction in multipoint crosses. Our results are consistent with a linear linkage group that shows positive chiasma interference and no chromatid interference. Chiasma interference occurs both within arms and across the centromere. Of the original loci that contributed to the circular map, we find that two map to other linkage groups and a third cannot be retested because the mutant strain that defined it has been lost. A second reported unusual property for linkage group XIX was the increase in meiotic recombination with increases in temperature during a period that precedes the onset of meiosis. Although we observed changes in recombination frequencies in some intervals on linkage group XIX in crosses to CC-1952, and in strains heterozygous for the mutation ger1 at 16°, we also show that our strains do not exhibit the previously observed patterns of temperature-sensitive recombination for two different pairs of loci on linkage group XIX. We conclude that linkage group XIX has a linear genetic map that is not significantly different from other Chlamydomonas linkage groups.     

Genetic recombination events which position the friedreich ataxia locus proximal to the D9S15/D9S5 linkage group on chromosome 9q

The absence of recombination between the mutation causing Friedreich ataxia and the two loci which originally assigned the disease locus to chromosome 9 has slowed attempts to isolate and characterize the genetic defect underlying this neurodegenerative disorder. A proximity of less than 1 cM to the linkage group has been proved by the generation of high maximal lod score (Z) to each of the two tightly linked markers D9S15 (Z = 96.69; recombination fraction [θ] = .01) and D9S5 (Z = 98.22; θ = .01). We report here recombination events which indicate that the FRDA locus is located centromeric to the D9S15/D9S5 linkage group, with the most probable order being cen–FRDA–D9S5–D9S15–qter. However, orientation of the markers with respect to the centromere, critical to the positional cloning strategy, remains to be resolved definitively.     

Cloning and characterization of centromeric DNA from Neurospora crassa.

The centromere locus from linkage group VII of Neurospora crassa has been cloned, characterized, and physically mapped. The centromeric DNA is contained within a 450-kb region that is recombination deficient, A+T-rich, and contains repetitive sequences. Repetitive sequences from within this region hybridize to a family of repeats located at or near centromeres in all seven linkage groups of N. crassa. Genomic Southern blots and sequence analysis of these repeats revealed a unique centromere structure containing a divergent family of centromere-specific repeats. The predominantly transitional differences between copies of the centromere-specific sequence repeats and their high A+T content suggest that their divergence was mediated by repeat-induced point (RIP) mutations.     

Genetic Positioning of Centromeres through Half-Tetrad Analysis in Gynogenetic Diploid Families of the Zhikong Scallop (Chlamys farreri)

Centromere mapping is a powerful tool for improving linkage maps, investigating crossover events, and understanding chiasma interference during meiosis. Ninety microsatellite markers selected across all linkage groups (LGs) from a previous Chlamys farreri genetic map were studied in three artificially induced meiogynogenetic families for centromere mapping by half-tetrad analysis. Inheritance analyses showed that all 90 microsatellite loci conformed to Mendelian inheritance in the control crosses, while 4.4 % of the microsatellite loci showed segregation departures from an expected 1:1 ratio of two homozygote classes in meiogynogenetic progeny. The second division segregation frequency (y) of the microsatellites ranged from 0.033 to 0.778 with a mean of 0.332, confirming the occurrence of partial chiasma interference in this species. Heterogeneity of y is observed in one of 42 cases in which markers were typed in more than one family, suggesting variation in gene–centromere recombination among families. Centromere location was mostly in accordance with the C. farreri karyotype, but differences in marker order between linkage and centromere maps occurred. Overall, this study makes the genetic linkage map a more complete and informative tool for genomic studies and it will also facilitate future research of the structure and function of the scallop centromeres.     

Half-Tetrad Analysis in Zebrafish: Mapping the Ros Mutation and the Centromere of Linkage Group I

Analysis of meiotic tetrads is routinely used to determine genetic linkage in various fungi. Here we apply tetrad analysis to the study of genetic linkage in a vertebrate. The half-tetrad genotypes of gynogenetic diploid zebrafish produced by early-pressure (EP) treatment were used to investigate the linkage relationships of two recessive pigment pattern mutations, leopard (leo) and rose (ros). The results showed that ros is tightly linked to its centromere and leo maps 31 cM from its centromere. Analysis of half-tetrads segregating for ros and leo in repulsion revealed no homozygous ros individuals among 32 homozygous leo half-tetrads--i.e., a parental ditype (PD) to nonparental ditype (NPD) ratio of 32:0. This result shows that ros is linked to leo, a mutation previously mapped to Linkage Group I. Investigation of PCR-based DNA polymorphisms on Linkage Group I confirmed the location of ros near the centromere of this linkage group. We propose an efficient, generally useful method to assign new mutations to a linkage group in zebrafish by determining which of 25 polymerase chain reaction (PCR)-based centromere markers shows a significant excess of PD to NPD in half-tetrad fish.     

Functional analysis of a centromere from fission yeast: a role for centromere-specific repeated DNA sequences.

A circular minichromosome carrying functional centromere sequences (cen2) from Schizosaccharomyces pombe chromosome II behaves as a stable, independent genetic linkage group in S. pombe. The cen2 region was found to be organized into four large tandemly repeated sequence units which span over 80 kilobase pairs (kb) of untranscribed DNA. Two of these units occurred in a 31-kb inverted repeat that flanked a 7-kb central core of nonhomology. The inverted repeat region had centromere function, but neither the central core alone nor one arm of the inverted repeat was functional. Deletion of a portion of the repeated sequences that flank the central core had no effect on mitotic segregation functions or on meiotic segregation of a minichromosome to two of the four haploid progeny, but drastically impaired centromere-mediated maintenance of sister chromatid attachment in meiosis I. This requirement for centromere-specific repeated sequences could not be satisfied by introduction of random DNA sequences. These observations suggest a function for the heterochromatic repeated DNA sequences found in the centromere regions of higher eucaryotes.     

Cytological Identification of the Chromosomes Involved in Searle''s Translocation and the Location of the Centromere in the X Chromosome of the Mouse

The autosome in Searle's X-autosome translocation has been shown to be chromosome 16. The breakpoint in chromosome 16 is slightly proximal to the middle and in the X is slightly distal to the middle.-Available evidence indicates that either Linkage Group XV or Linkage Group XIX is carried on chromosome 16.-The centromere of the X chromosome is at the spf end of the linkage group.     

Microheterogeneity of the inner core region of yeast manno-protein

The inner core linkage region fragment from Saccharomyces cerevisiae mannan has been fractionated into 6 components and their structures have been analyzed. They form a family of homologous oligosaccharides (Man₁₂GNAc to Man₁₇GNAc) with 6 or 7 mannose units in α1→6 linkage attached to N-acetylglucosamine by a β1→4 linkage, and with different amounts of side chain mannose units attached by α1→2 and α1→3 linkage.     

Redistribution of centrosomal proteins by centromeres and Polo kinase controls partial nuclear envelope breakdown in fission yeast

Proper mitotic progression in Schizosaccharomyces pombe requires partial nuclear envelope breakdown (NEBD) and insertion of the spindle pole body (SPB—yeast centrosome) to build the mitotic spindle. Linkage of the centromere to the SPB is vital to this process, but why that linkage is important is not well understood. Utilizing high-resolution structured illumination microscopy, we show that the conserved Sad1-UNC-84 homology-domain protein Sad1 and other SPB proteins redistribute during mitosis to form a ring complex around SPBs, which is a precursor for localized NEBD and spindle formation. Although the Polo kinase Plo1 is not necessary for Sad1 redistribution, it localizes to the SPB region connected to the centromere, and its activity is vital for redistribution of other SPB ring proteins and for complete NEBD at the SPB to allow for SPB insertion. Our results lead to a model in which centromere linkage to the SPB drives redistribution of Sad1 and Plo1 activation that in turn facilitate partial NEBD and spindle formation through building of a SPB ring structure.     

A Genetic linkage map of Atlantic halibut (Hippoglossus hippoglossus L.)

A genetic linkage map has been constructed for Atlantic halibut on the basis of 258 microsatellites and 346 AFLPs. Twenty-four linkage groups were identified, consistent with the 24 chromosomes seen in chromosome spreads. The total map distance is 1562.2 cM in the female and 1459.6 cM in the male with an average resolution of 4.3 and 3.5 cM, respectively. Using diploid gynogens, we estimated centromere locations in 19 of 24 linkage groups. Overall recombination in the female was approximately twice that of the male; however, this trend was not consistent along the linkage groups. In the centromeric regions, females had 11-17.5 times the recombination of the males, whereas this trend reversed toward the distal end with males having three times the recombination of the females. Correspondingly, in the male, markers clustered toward the centromeric region with 50% of markers within 20 cM of the putative centromere, whereas 35% of markers in the female were found between 60 and 80 cM from the putative centromere. Limited interspecies comparisons within Japanese flounder and Tetraodon nigroviridis revealed blocks of conservation in sequence and marker order, although regions of chromosomal rearrangement were also apparent.     

Studies on the linkage map of chromosome 4 of the tomato and on the transmission of induced deficiencies

Various genetic and cytogenetic techniques were applied to an analysis of the linkage map of chromosome 4-a chromosome that is considered to be representative of the tomato complement. Loci have been approximated by standard F₂ linkage tests for 18 genes, including six on the short arm and 12 on the long arm, covering a map distance of 132 units (c.m.). The loci of four key markers were approximated on pachytene chromosomes by a study of radiation-induced deficiencies:clau near the end of the short arm,ful near the euchromatic-heterochromatic boundary of the short arm,ra near the same region on the long arm, ande in the middle of the long arm. Normal transmission for a presumedra deficiency suggests that this gene lies in the heterochromatin of 4L. According to tertiary trisomic segregation,w-4, known by linkage test to be proximal tora, resides on 4L, therefore probably also in the heterochromatic region. The centromere is consequently delimited to a region of 4 c.m. betweenful andw-4. The resultant maps reveal a very much lower crossover rate within heterochromatin—estimated at 0.8 c.m./μ—than for euchromatin—estimated at 4.8 c.m./μ for the short arm and 5.7 for the long arm. Also apparent is a strong tendency of the genes to concentrate toward the centromere of the genetic map and in the proximal sections of the euchromatin of the cytological map. Studies were made of the genetic transmission of various small deficiencies on chromosome 4 as well as a newly discovered deficiency fornv on chromosome 9, supporting the following conclusions. Regardless of their size, deficiendies of euchromatin are not transmitted. Deficiencies of heterochromatin are transmitted to a varying extent depending on their size. A presumed deficiency forra that is too small to be detected cytologically was transmitted without adverse effect on gametes. Somewhat larger deficiencies may be transmitted at reduced rates by female gametes and the largest at extremely low rates, even on the female side.     

Recessive Lethal Mutations and the Maintenance of Duplication-Bearing Strains of Dictyostelium discoideum

Recessive lethal mutations have been isolated and used to maintain n + 1 aneuploid strains of Dictyostelium discoideum carrying a duplication of part or all of linkage group VII. The recessive lethal mutations, relA351 and relB352, arose spontaneously in diploids; no mutagenic treatment was used in the isolation of these mutations. The probable gene order on linkage group VII is: centromere, relB couA, bsgB, cobA, relA. Maintenance of aneuploids disomic for linkage group VII was made possible by complementation of a rel mutation on each linkage group VII homologue by the corresponding wild-type allele on the other linkage group VII homologue. The duplication-bearing disomic strains were slow-growing and produced faster-growing sectors on the colony edge. Haploid sectors probably arise by a combination of mitotic recombination and subsequent loss of one homologue, diploid sectors may be formed by chromosome doubling to 2n + 2, followed by chromosome loss to return to 2n, and aneuploid sectors may arise by deletion or new mutation.     

Linkage studies involving two chromosomal male-sterility mutants in hexaploid wheat

The factors for Cornerstone and Probus male sterility are allelic on chromosome arm 4Aα. They map independently of the centromere, but show linkage with a rye segment located 1 crossover unit from the centromere on the β arm. The alien segment causes asynapsis and some precocious terminalization of chiasmata when in repulsion with the mutants. The mutants, presumed to be terminal deletions, cause some desynapsis, but not asynapsis.     

Meiotic drive in fungi: Chromosomal elements that cause fratricide and distort genetic ratios

Fungal Spore killers (Sk), studied most extensively inNeurospora and to a lesser extent inPodospora, Gibberella andCochliobolus, cause the death of ascospores (= meiospores) that do not contain the killer (Skk) element. When a Spore killer is heterozygous (SkK× Sks) inNeurospora, every ascus (= meiocyte) contains four normal-sized, black, viable ascospores (SkK), and four ascospores that are tiny, unpigmented and unviable (SKs). Killing of sensitive nuclei is expressed postmeiotically, and results in gross distortion of segregation ratios forSk-linked genes. A sensitive nucleus that would otherwise die is rescued if a killer nucleus is also enclosed in the same ascospore. InNeurospora, Sk is centromere-linked (linkage group III), and when heterozygous, shows a recombination block in a 30-map-unit region spanning the centromere of linkage group III. There is no ascospore death or recombination block in killer×killer or sensitive×sensitive crosses. Spore killers are fairly common inGibberella fujikuroi andNeurospora sitophila but extremely rare inN. intermedia, and have not yet been found among natural isolates ofN. crassa.     

An integrated genetic and physical map of the bovine X Chromosome

Genotypic data for 56 microsatellites (ms) generated from maternal full sib families nested within paternal half sib pedigrees were used to construct a linkage map of the bovine X Chromosome (Chr) (BTX) that spans 150 cM (ave. interval 2.7 cM). The linkage map contains 36 previously unlinked ms; seven generated from a BTXp library. Genotypic data from these 36 ms was merged into an existing linkage map to more than double the number of informative BTX markers. A male specific linkage map of the pseudoautosomal region was also constructed from five ms at the distal end of BTXq. Four informative probes physically assigned by fluorescence in situ hybridization defined the extent of coverage, confirmed the position of the pseudoautosomal region on the q-arm, and identified a 4.1-cM marker interval containing the centromere of BTX. Received: 14 July 1996 / Accepted: 19 September 1996     

Centromere locations and associated chromosome rearrangements in Arabidopsis lyrata and A. thaliana

We analyzed linkage and chromosomal positions of genes in A. lyrata ssp. petraea that are located near the centromere (CEN) regions of A. thaliana, using at least two genes from the short and long arms of each chromosome. In our map, genes from all 10 A. thaliana chromosome arms are also tightly linked in A. lyrata. Genes from the regions on the two sides of CEN5 have distant map localizations in A. lyrata (genes on the A. thaliana short-arm genes are on linkage group AL6, and long-arm genes are on AL7), but genes from the other four A. thaliana centromere regions remain closely linked in A. lyrata. The observation of complete linkage between short- and long-arm centromere genes, but not between genes in other genome regions that are separated by similar physical distances, suggests that crossing-over frequencies near the A. lyrata ssp. petraea centromere regions are low, as in A. thaliana. Thus, the centromere positions appear to be conserved between A. thaliana and A. lyrata, even though three centromeres have been lost in A. thaliana, and the core satellite sequences in the two species are very different. We can now definitively identify the three centromeres that were eliminated in the fusions that formed the A. thaliana chromosomes. However, we cannot tell whether genes were lost along with these centromeres, because such genes are absent from the A. thaliana genome, which is the sole source of markers for our mapping.     

Frameshift Suppression in SACCHAROMYCES CEREVISIAE. III. Isolation and Genetic Properties of Group III Suppressors

Suppressors of ICR-induced mutations that exhibit behavior similar to bacterial frameshift suppressors have been identified in the yeast Saccharomyces cerevisiae. The yeast suppressors have been divided into two groups. Previous evidence indicated that suppressors of one group (Group II: SUF1, SUF3, SUF4, SUF5 and SUF6) represent mutations in the structural genes for glycyl-tRNA's. Suppressors of the other group (Group III: SUF2 and SUF7) were less well characterized. Although they suppressed some ICR-revertible mutations, they failed to suppress Group II frameshift mutations. This communication provides a more thorough characterization of the Group III suppressors and describes the isolation and properties of four new suppressors in that group (SUF8, SUF9, SUF10 and suf11).——In our original study, Group III suppressors were isolated as revertants of the Group III mutations his4–712 and his4–713. All suppressors obtained as ICR-induced revertants of these mutations mapped at the SUF2 locus near the centromere of chromosome III. Suppressors mapping at other loci were obtained in this study by analyzing spontaneous and UV-induced revertants of the Group III mutations. SUF2 and SUF10 suppress both Group III his4 mutations, whereas SUF7, SUF8, SUF9 and suf11 suppress his4–713, but not his4–712. All of the suppressors except suf11 are dominant in diploids homozygous for his4-713. The suppressors fail to suppress representative UAA, UAG and UGA nonsense mutations.——SUF9 is linked to the centromere of chromosome VI, and SUF10 is linked to the centromere of chromosome XIV. A triploid mapping procedure was used to determine the chromosome locations of SUF7 and SUF8. Subsequent standard crosses revealed linkage of SUF7 to cdc5 on chromosome XIII and linkage of SUF8 to cdc12 and pet3 on chromosome VIII.