Haploid Progeny Research Articles

The influence of temperature on chromosome elimination during embryo development in crosses involving Hordeum spp., wheat (Triticum aestivum L.) and rye (Secale cereale L.)

Several interspecific and intergeneric crosses involving five Hordeum species, Triticum aestivum and Secale cereale were carried out to investigate the influence of two contrasting temperatures on chromosome elimination during embryo development. In four of the interspecific Hordeum crosses, chromosome elimination was significantly increased at the higher of the two temperatures, resulting in greater proportions of haploid plant progenies. However, there was no significant effect of temperature in the other interspecific cross between H. lechleri x H. bulbosum nor in the two intergeneric crosses between H. vulgare x S. cereale and T. aestivum x H. bulbosum whose progeny were exclusively hybrid and haploid, respectively.

Functional homology of mammalian and yeast RAS genes

Yeast spores lacking endogenous RAS genes will not germinate. If such spores contain chimeric mammalian/yeast RAS genes or even the mammalian H- ras gene under the control of the galactose-inducible GAL10 promoter, they will germinate in the presence of galactose and produce viable haploid progeny dependent on galactose for continued growth and viability. These results indicate that the biochemical function of RAS proteins is essential for vegetative haploid yeast and that this function has been conserved in evolution since the progenitors of yeast and mammals diverged.

Development of Rapidly Fermenting Strains of Saccharomyces diastaticus for Direct Conversion of Starch and Dextrins to Ethanol

Alcoholic fermentation, growth, and glucoamylase production by 12 strains of Saccharomyces diastaticus were compared by using starch and dextrins as substrates. Haploid progeny produced from a rapidly fermenting strain, SD2, were used for hybridization with other S. diastaticus and Saccharomyces cerevisiae haploids. Alcoholic fermentation and enzyme production by hybrid diploids and their haploid parents were evaluated. Although the dosage of the STA or DEX (starch or dextrin fermentation) genes may enhance ethanol production, epistatic effects in certain strain combinations caused decreases in starch-fermenting activity. Both the nature of the starch or dextrin used and the fermentation medium pH had substantial effects on alcohol production. Commercial dextrin was not as good a substrate as dextrins prepared by digesting starch with alpha-amylase. Crude manioc starch digested by alpha-amylase was fermented directly by selected hybrids with almost 100% conversion efficiency. The manioc preparation contained adequate minerals and growth factors. This procedure should be suitable for direct commercial application in manioc-producing regions in Brazil and elsewhere. A rapidly fermenting haploid strain, SD2-A8, descended from strain SD2, contains two unlinked genes controlling formation of extracellular amylase. A convenient method for detecting these genes (STA genes) in replica plates containing large numbers of meiotic progeny was developed.

Assessment of mutagenic damage by monofunctional alkylating agents and gamma radiation in haploid and diploid frogs, Xenopus laevis.

Adult male South African clawed frogs, Xenopus laevis, were mutagenized by 3-day immersion in aqueous solutions of ethyl methanesulfonate (EMS), diethyl nitrosamine (DEN), or ethyl nitrosourea (ENU), or by acute exposure to gamma radiation. They were then spawned repeatedly at 2-week intervals with untreated females, and embryonic survival of the progeny was used to assess genetic damage. Recessive lethal effects were assessed from reduced survival of androgenetic haploid progeny. Neither recessive nor dominant lethal effects were obtained after exposure to 100 mg/liter EMS or 2 g/liter DEN. At 250 mg/liter EMS, peak dominant lethality occurred 3-5 wk after treatment. Most embryos hatched, but many were abnormal and died shortly after hatching. Haploid survival was significantly reduced over a broader period, from 1 to 13 wk after mutagenesis. Treatment with 75 mg/liter ENU produced effects similar to the 250-mg/liter EMS mutagenesis. At 400 mg/liter EMS, the frequency and severity of the effects on both diploid and haploid embryos were increased over the lower dose. Gamma irradiation at 1,500 R produced effects similar to the 400-mg/liter mutagenesis, except that peak dominant lethality extended from 1 to 7 wk.

Test for temporal or spatial restrictions in gene product function during the cell division cycle.

The ability of a functional gene to complement a nonfunctional gene may depend upon the intracellular relationship of the two genes. If so, the function of the gene product in question must be limited in time or in space. CDC (cell division cycle) gene products of Saccharomyces cerevisiae control discrete steps in cell division; therefore, they constitute reasonable candidates for genes that function with temporal or spatial restrictions. In an attempt to reveal such restrictions, we compared the ability of a CDC gene to complement a temperature-sensitive cdc gene in diploids where the genes are located within the same nucleus to complementation in heterokaryons where the genes are located in different nuclei. In CDC X cdc matings, complementation was monitored in rare heterokaryons by assaying the production of cdc haploid progeny (cytoductants) at the restrictive temperature. The production of cdc cytoductants indicates that the cdc nucleus was able to complete cell division at the restrictive temperature and implies that the CDC gene product was provided by the other nucleus or by cytoplasm in the heterokaryon. Cytoductants from cdc28 or cdc37 crosses were not efficiently produced, suggesting that these two genes are restricted spatially or temporally in their function. We found that of the cdc mutants tested 33 were complemented; cdc cytoductants were recovered at least as frequently as CDC cytoductants. A particularly interesting example was provided by the CDC4 gene. Mutations in CDC4 were found previously to produce a defect in both cell division and karyogamy. Surprisingly, the cell division defect of cdc4 nuclei is complemented by CDC4 nuclei in a heterokaryon, whereas the karyogamy defect is not.

Inheritance of extrachromosomal rDNA in Physarum polycephalum.

In the acellular slime mold Physarum polycephalum, the several hundred genes coding for rRNA are located on linear extrachromosomal DNA molecules of a discrete size, 60 kilobases. Each molecule contains two genes that are arranged in a palindromic fashion and separated by a central spacer region. We investigated how rDNA is inherited after meiosis. Two Physarum amoebal strains, each with an rDNA recognizable by its restriction endonuclease cleavage pattern, were mated, the resulting diploid plasmodium was induced to sporulate, and haploid progeny clones were isolated from the germinated spores. The type of rDNA in each was analyzed by blotting hybridization, with cloned rDNA sequences used as probes. This analysis showed that rDNA was inherited in an all-or-nothing fashion; that is, progeny clones contained one or the other parental rDNA type, but not both. However, the rDNA did not segregate in a simple Mendelian way; one rDNA type was inherited more frequently than the other. The same rDNA type was also in excess in the diploid plasmodium before meiosis, and the relative proportions of the two rDNAs changed after continued plasmodial growth. The proportion of the two rDNA types in the population of progeny clones reflected the proportion in the parent plasmodium before meoisis. The rDNAs in many of the progeny clones contained specific deletions of some of the inverted repeat sequences at the central palindromic symmetry axis. To explain the pattern of inheritance of Physarum rDNA, we postulate that a single copy of rDNA is inserted into each spore or is selectively replicated after meiosis.

Inheritance of extrachromosomal rDNA in Physarum polycephalum.

In the acellular slime mold Physarum polycephalum, the several hundred genes coding for rRNA are located on linear extrachromosomal DNA molecules of a discrete size, 60 kilobases. Each molecule contains two genes that are arranged in a palindromic fashion and separated by a central spacer region. We investigated how rDNA is inherited after meiosis. Two Physarum amoebal strains, each with an rDNA recognizable by its restriction endonuclease cleavage pattern, were mated, the resulting diploid plasmodium was induced to sporulate, and haploid progeny clones were isolated from the germinated spores. The type of rDNA in each was analyzed by blotting hybridization, with cloned rDNA sequences used as probes. This analysis showed that rDNA was inherited in an all-or-nothing fashion; that is, progeny clones contained one or the other parental rDNA type, but not both. However, the rDNA did not segregate in a simple Mendelian way; one rDNA type was inherited more frequently than the other. The same rDNA type was also in excess in the diploid plasmodium before meiosis, and the relative proportions of the two rDNAs changed after continued plasmodial growth. The proportion of the two rDNA types in the population of progeny clones reflected the proportion in the parent plasmodium before meoisis. The rDNAs in many of the progeny clones contained specific deletions of some of the inverted repeat sequences at the central palindromic symmetry axis. To explain the pattern of inheritance of Physarum rDNA, we postulate that a single copy of rDNA is inserted into each spore or is selectively replicated after meiosis.

Gynogenesis caused by ultraviolet irradiation of salmonid sperm.

Gynogenetic rainbow trout lines can be produced easily with the simple UV sperm irradiation technique detailed in this study. The dose effect on embryonic survival rate is called a pseudo "Hertwig effect" because of major differences with results of gamma irradiation: Some mixing of various karyotypes (from diploid to haploid number) were obtained with mean doses, probably because of the weak penetrating power of the ultraviolet rays. Irradiations lasting 4 minutes of more are required to produce homogeneous haploid progeny; heat shock applied to such fertilized eggs results in diploid fry of the maternal phenotype.

THE ROLE OF S. CEREVISIAE CELL DIVISION CYCLE GENES IN NUCLEAR FUSION

Forty temperature-sensitive cell division cycle (cdc) mutants of Saccharomyces cerevisiae were examined for their ability to complete nuclear fusion during conjugation in crosses to a CDC parent strain at the restrictive temperature. Most of the cdc mutant alleles behaved as the CDC parent strain from which they were derived, in that zygotes produced predominantly diploid progeny with only a small fraction of zygotes giving rise to haploid progeny (cytoductants) that signalled a failure in nuclear fusion. However, cdc4 mutants exhibited a strong nuclear fusion (karyogamy) defect in crosses to a CDC parent and cdc28, cdc34 and cdc37 mutants exhibited a weak karyogamy defect. For all four mutants, the karyogamy defect and the cell cycle defect cosegregated, suggesting that both defects resulted from a single lesion for each of these cdc mutants. Therefore, the cdc 4, 28, 34 and 37 gene products are required in both cell division and karyogamy.

Internuclear transfer of genetic information in kar1-1/KAR1 heterokaryons in Saccharomyces cerevisiae.

Heterokaryons of Saccharomyces cerevisiae have been constructed utilizing the kar1-1 mutation, which prevents nuclear fusion during conjugation (J. Conde and G. Fink, Proc. Natl. Acad. Sci. U.S.A. 73:3651-3655, 1976). Each heterokaryon contained two haploid nuclei that were marked on several chromosomes. They segregated haploid progeny (cytoductants), most of which have the nuclear genotype of one or the other of the heterokaryon parents, but they occasionally segregated progeny having a recombinant genotype (exceptional cytoductants). Exceptional cytoductants receive the majority of their genome from one parent (the recipient) and a minority from the other (the donor). Transfer of two markers from the donor nucleus to the recipient is rarely coincident for markers located on different chromosomes but is nearly always coincident for those markers located on the same chromosome, suggesting that whole chromosomes are transferred from the donor nucleus to the recipient. In crosses of kar1-1 X KAR1 parents, either nucleus may act as a recipient or donor with equal probability. Recipient nuclei acquired 9 of the 10 chromosomes examined, with frequencies which were inversely correlated with the size of the chromosome. When a chromosome is acquired by the recipient nucleus, it either replaces its homolog or exists in a disomic condition. Haploid progeny emanating from kar1 X KAR1 crosses are frequently inviable. I tested whether this inviability might be the result of chromosome loss by donor nuclei. Viability of progeny from kar1 X KAR1 heterokaryons was improved when the parental nuclei were diploid to an extent consistent with the hypothesis, and diploid progeny which had become monosomic were recovered from these heterokaryons. The following sequence of events accounts for chromosome transfer in kar1 X KAR1 heterokaryons. After cell fusion, each nucleus in the heterokaryon has a probability of about 0.38 of losing one or more chromosomes. A nucleus sustaining such a loss can become a donor in a chromosome transfer event. If the other nucleus does not sustain a mortal chromosome loss, it can become a recipient in a transfer event. The chance of acquiring a chromosome lost by the donor is greater for smaller chromosomes than for larger ones and is about 0.05 for the average chromosome.

Internuclear transfer of genetic information in kar1-1/KAR1 heterokaryons in Saccharomyces cerevisiae.

Heterokaryons of Saccharomyces cerevisiae have been constructed utilizing the kar1-1 mutation, which prevents nuclear fusion during conjugation (J. Conde and G. Fink, Proc. Natl. Acad. Sci. U.S.A. 73:3651-3655, 1976). Each heterokaryon contained two haploid nuclei that were marked on several chromosomes. They segregated haploid progeny (cytoductants), most of which have the nuclear genotype of one or the other of the heterokaryon parents, but they occasionally segregated progeny having a recombinant genotype (exceptional cytoductants). Exceptional cytoductants receive the majority of their genome from one parent (the recipient) and a minority from the other (the donor). Transfer of two markers from the donor nucleus to the recipient is rarely coincident for markers located on different chromosomes but is nearly always coincident for those markers located on the same chromosome, suggesting that whole chromosomes are transferred from the donor nucleus to the recipient. In crosses of kar1-1 X KAR1 parents, either nucleus may act as a recipient or donor with equal probability. Recipient nuclei acquired 9 of the 10 chromosomes examined, with frequencies which were inversely correlated with the size of the chromosome. When a chromosome is acquired by the recipient nucleus, it either replaces its homolog or exists in a disomic condition. Haploid progeny emanating from kar1 X KAR1 crosses are frequently inviable. I tested whether this inviability might be the result of chromosome loss by donor nuclei. Viability of progeny from kar1 X KAR1 heterokaryons was improved when the parental nuclei were diploid to an extent consistent with the hypothesis, and diploid progeny which had become monosomic were recovered from these heterokaryons. The following sequence of events accounts for chromosome transfer in kar1 X KAR1 heterokaryons. After cell fusion, each nucleus in the heterokaryon has a probability of about 0.38 of losing one or more chromosomes. A nucleus sustaining such a loss can become a donor in a chromosome transfer event. If the other nucleus does not sustain a mortal chromosome loss, it can become a recipient in a transfer event. The chance of acquiring a chromosome lost by the donor is greater for smaller chromosomes than for larger ones and is about 0.05 for the average chromosome.

The genetic analysis of barley storage proteins

Hordein polypeptide patterns in barley seeds are known to be controlled by structural genes at 2 loci, Hor-1 and Hor-2, on chromosome 5. Two-dimensional and high resolution one-dimensional electrophoretic analyses of seeds of F2 and doubled haploid progenies of four intervarietal crosses gave no evidence of recombination within these loci. Genetic analysis of the progenies showed that Hor-1 is 0·161 + 0·026 centimorgans from Hor-2 and the mildew resistance locus, Mla, lies between them, 0·064 ± 0·020 centimorgans from Hor-1 and 0·082 + 0·024 centimorgans from Hor-2.

Genetic evidence for nuclear location of 2-micron DNA in yeast

Progeny fromkarl×KAR1 crosses in Saccharomyces cerevisiae were isolated by selection for a mitochondrial marker from each parent. Haploid progeny (heteroplasmons, cytoductants) were obtained at high frequency because of thekar mutation and were identified by replication of progeny colonies to media differentiating on the basis of chromosomal markers. In some of the crosses nuclei of both parental types were found among the progeny, although theKAR1 nuclei were in majority.In crosses where only one parent contained the plasmid 2-micron DNA, all progeny inheriting the nucleus from that parent also had the plasmid, whereas only about 25–50% of the progeny with the other nucleus had the plasmid. In crosses where the size of 2-micron DNA of the parents was different, the haploid progeny had a plasmid size identical to that of the parent from which it had received its nucleus.On the basis of these observations we suggest a nuclear location of 2-micron DNA. We propose that one or a few copies of the plasmid are frequently transferred from one nucleus to another inkar1 mediated transient heterokaryons.

Gene Expression in Haploid and Hybrid Progeny from Crosses Between Hordeum vulgare and H. bulbosum1

Gene expression was examined in haploid and hybrid progeny resulting from interspecific crosses of diploid Hordeum vulgare L. genetic marker stocks with diploid and tetraploid forms of H. bulbosum L. The expression of marker genes on all vulgare chromosomes occurred in the haploid progeny, an observation consistent with the concept that the haploids contain only vulgare chromosomes and that bulbosum chromosomes are selectively eliminated. However, the recessive genes of H. vulgare were masked in diploid and triploid hybrids by H. bulbosum genes, which is indicative of the occurrence of interallelic interactions and genome homology between the species. Gene interaction and expression in interspecific and intergeneric hybrids in relation to alien gene or chromosome transfers is discussed.

Identification of Genes at the Ml‐a Locus in Barley for Resistance to Erysiphe graminis F. sp. Hordei1

The pathogenicity of haploid progeny from the cross of cultures CR3 and 63.5 of Erysiphe graminis [DC.] Merat hordei Em. Marchal was determined on 11 barley (Hordeum vulgare L.) varieties, each known to have a resistance gene at the Ml‐a locus. Different genes were shown to condition the pathogenicity of cultures CR3 and 63.5 on the ll varieties, indicating the corresponding resistance genes in those varieties are different. Genes in seven of the varieties — ‘Algerian,’ ‘Black Russian,’ ‘Ricardo,’ ‘No. 22,’ ‘Gopal,’ ‘Iso 20,’ and ‘Line 4831’ — had previously been designated Ml‐a through Ml‐a7. We propose the genes in varieties ‘Heil's Hanna,’ ‘Monte Cristo,’ ‘Iso 12,’ and ‘A222’ be designated Ml‐a8, Ml‐a9, Ml‐al0, and Ml‐a11, respectively.

Congenital Body Malformations and Eye Color Mutations in Progeny from Irradiated Female Wasps (Dahlbominus)

Body malformations occurring in haploid male progeny from irradiated Dahlbominus females are described, and the rises in frequencies with advancing maternal age and with chronic versus acute exposures are shown to resemble the corresponding relationships for eye color mutations. This similarity in the two types of change, together with tests of the heritability of those malformations which were viable, indicate that most of the induced body anomalies are mutational in origin. This evidence from insects may be important for the interpretation of radiation effects in man, since insect embryos are not subject to prenatal accidents such as failures to implant and abortions, and thus can develop at least to the larval or the pupal stages even when the individual is abnormal. The rise in the frequencies with advancing age is known to be due to an increase in the number of radiation-sensitive oocytes in the ovarioles.

A Quarter Century Exploring Tetrahymena*

SYNOPSIS. In reviewing 25 years of work on Tetrahymena, the author describes early difficulties experienced by himself and others in developing a chemically‐defined medium and in defining the taxonomy of T. pyriformis. With identification of its essential requirements, a variety of nutritional studies revealed the close resemblance between the metabolic pathways of this ciliate and those of the chick and rat. This suggested studies on the effects of gamma radiation and the effect of antibiotics.Attention then turned to the puzzle of the absence of conjugation in all strains of T. pyriformis then maintained in laboratories. The observation that these strains were all amicronucleate led to the discovery of mating types (with D. F. Gruchy). This brought up the problem of what constitutes a species and how it might be solved in T. pyriformis where clearly defined groups are set off from other similar groups (varieties) by a breeding barrier.Another problem is whether a life cycle exists in this ciliate and if so, the role amicronucleate strains play in this cycle.The origin and obvious evolutionary success of these “genetically dead” strains are other problems that are considered.The search for nutritional markers for genetic experiments inspired large‐scale collections, including some from tropical habitats. Strains were found which differed in serine and pyridoxine requirements. Genetic analysis was hindered by the high lethality following conjugation, unsatisfactory mating behavior in some clones, and the complete absence of autogamy, the latter making it difficult to obtain homozygous clones. The use of x‐radiation yielded haploid individuals; when these were mated with diploid clones the resulting haploid progeny proved that the genes controlling mating type and pyridoxine synthesis resided in the micronucleus.Genetic analysis of the serine and pyridoxine mutants supports the idea that T. pyriformis is more of an outbreeder than an inbreeder. Left for the future is an extension of our knowledge of the breeding system of T. pyriformis and ultimately to examine critically the factors responsible for the success of this ubiquitous ciliate.

'Long-Term’ Adaptation and Non-Mendelian Inheritance in Yeast

HAPLOPHASE yeasts capable of fermenting galactose ordinarily produce gas in from sixteen to thirty-six hours at 30° C. after inoculation into nutrient medium containing galactose. Winge and Roberts1 studied clones which fermented the sugar in from three to fourteen days after they were seeded in galactose yeastants to the time required by the cells in the haploid popu extract medium. Following the adaptation period, however, the rate of fermentation was identical with that of ordinary galactose-fermenting yeasts. In their analyses, they found that two haploid segregants from asci heterozygous for galactose fermentation were the ordinary fast fermenters and the other two were ‘long-term’ adapting clones. They attributed the delay in fermentation of the ‘long-term’ adapting segreglation to build up galactozymase to a concentration sufficient for the detection of its presence. The regular segregations of fast/'long-term’ adapting phenotypes among the haploid progeny were offered as proof that delayed fermentation of galactose is controlled by a specific allele. Such originally 2: 2 fast/'long-term’ tetrads were transformed into 4: 0 fast/'long-term’ ratios by extended cultivation in galactose yeast extract medium. Winge and Roberts considered that under these conditions there was no change in the population but merely a ‘long-term’ adaptation of the entire population. In proposing this view, they denied the occurrence of mutations as a factor operative in the ‘long-term’ adaptation, and held that de-adaptation of an adapted clone, as well as its subsequent re-adaptation to galactose, were effected through a modification.