Genetic basis of the major malate dehydrogenase isozymes in maize

The mitochondrial MDH isozymes in the scutellum of the mature maize (Zea mays L.) kernel are encoded by three independently inherited nuclear genes. Mdh1 is located on chromosome 8, close to the breakpoint (8L.35) of a waxy-marked reciprocal translocation between chromosomes 8 and 9. Mdh2 is located in the distal region of the long arm of chromosome 6. Mdh3 is on the long arm of chromosome 3, approximately 2.6 map units from sh2. A modifier of the mitochondrial MDH isozymes (Mmm) maps approximately 27.5 units proximal to Adh1 in the central portion of the long arm of chromosome 1. Independently assorting duplicate genes code for the soluble MDH isozymes. Mdh4 is located in the same region of chromosome 1 as Mmm, approximately 29 map units proximal to Adh1. Mdh5 maps approximately 20 units distal to a2 in the short arm of chromosome 5.——Intergenic and interallelic heterodimer formation occurs among gene products that occupy the same subcellular compartment. MDH isozymes were purified and analyzed by native-SDS two-dimensional polyacrylamide gel electrophoresis. The proposed mitochondrial MDH intergenic heterodimer bands were found to be composed of two subunits, which differ in their migrations on SDS gels; whereas, genetically defined homodimers contained only one type of subunit.——This evidence is discussed in terms of two genetic models proposed for the maize mitochondrial MDH isozymes.     

Association of Isozymes with a Reciprocal Translocation in Cultivated Rye (SECALE CEREALE L.)

In rye (Secale cereale L. cv. "Ailés") the progeny of a cross between a structural heterozygote for a reciprocal translocation (involving the 1R chromosome) and a homozygote for the standard chromosome arrangement were analyzed for the electrophoretic patterns of eight different leaf isozymes and also for their meiotic configuration at metaphase I.——The Got-3 and Mdh-2b loci are linked to each other and also to the reciprocal translocation. The Mdh-2b locus is located in the interstitial segment of the 3Rq chromosome arm, with an estimated distance of 8 cM to the breakpoint. Therefore, the reciprocal translocation involves the 1R and 3R chromosomes.——Also, the Mdh-1 and 6-Pgd-2 loci are linked (16 ± 3 cM) and have been located on the 2Rq arm. Finally, the Per-3 and Per-4 loci are located on the 2Rp chromosome arm at an estimated distance of 26 ± 4 cM.     

Two Chromosomal Genes Required for Killing Expression in Killer Strains of SACCHAROMYCES CEREVISIAE

The killer character of yeast is determined by a 1.4 x 10⁶ molecular weight double-stranded RNA plasmid and at least 12 chromosomal genes. Wild-type strains of yeast that carry this plasmid (killers) secrete a toxin which is lethal only to strains not carrying this plasmid (sensitives). ——— We have isolated 28 independent recessive chromosomal mutants of a killer strain that have lost the ability to secrete an active toxin but remain resistant to the effects of the toxin and continue to carry the complete cytoplasmic killer genome. These mutants define two complementation groups, kex1 and kex2. Kex1 is located on chromosome VII between ade5 and lys5. Kex2 is located on chromosome XIV, but it does not show meiotic linkage to any gene previously located on this chromosome. ——— When the killer plasmid of kex1 or kex2 strains is eliminated by curing with heat or cycloheximide, the strains become sensitive to killing. The mutant phenotype reappears among the meiotic segregants in a cross with a normal killer. Thus, the kex phenotype does not require an alteration of the killer plasmid. ——— Kex1 and kex2 strains each contain near-normal levels of the 1.4 x 10⁶ molecular weight double-stranded RNA, whose presence is correlated with the presence of the killer genome.     

Temperature-Sensitive Lethal Mutations on Yeast Chromosome I Appear to Define Only a Small Number of Genes

A method was developed for isolating large numbers of mutations on chromosome I of the yeast Saccharomyces cerevisiae. A strain monosomic for chromosome I (i.e., haploid for chromosome I and diploid for all other chromosomes) was mutagenized with either ethyl methanesulfonate or N-methyl-N'-nitro-N -nitrosoguanidine and screened for temperature-sensitive (Ts^- ) mutants capable of growth on rich, glucose-containing medium at 25° but not at 37°. Recessive mutations induced on chromosome I are expressed, whereas those on the diploid chromosomes are usually not expressed because of the presence of wild-type alleles on the homologous chromosomes. Dominant ts mutations on all chromosomes should also be expressed, but these appeared rarely. — Of the 41 ts mutations analyzed, 32 mapped on chromosome I. These 32 mutations fell into only three complementation groups, which proved to be the previously described genes CDC15, CDC24 and PYK1 (or CDC19). We recovered 16 or 17 independent mutations in CDC15, 12 independent mutations in CDC24 and three independent mutations in PYK1. A fourth gene on chromosome I, MAK16, is known to be capable of giving rise to a ts-lethal allele, but we recovered no mutations in this gene. The remaining nine mutations isolated using the monosomic strain appeared not to map on chromosome I and were apparently expressed in the original mutants because they had become homozygous or hemizygous by mitotic recombination or chromosome loss. — The available information about the size of chromosome I suggests that it should contain approximately 60–100 genes. However, our isolation in the monosomic strain of multiple, independent alleles of just three genes suggests that only a small proportion of the genes on chromosome I is easily mutable to give a Ts^--lethal phenotype. — During these studies, we located CDC24 on chromosome I and determined that it is centromere distal to PYK1 on the left arm of the chromosome.     

Candidate Gene Association Study in Type 2 Diabetes Indicates a Role for Genes Involved in β-Cell Function as Well as Insulin Action

Type 2 diabetes is an increasingly common, serious metabolic disorder with a substantial inherited component. It is characterised by defects in both insulin secretion and action. Progress in identification of specific genetic variants predisposing to the disease has been limited. To complement ongoing positional cloning efforts, we have undertaken a large-scale candidate gene association study. We examined 152 SNPs in 71 candidate genes for association with diabetes status and related phenotypes in 2,134 Caucasians in a case-control study and an independent quantitative trait (QT) cohort in the United Kingdom. Polymorphisms in five of 15 genes (33%) encoding molecules known to primarily influence pancreatic β-cell function—ABCC8 (sulphonylurea receptor), KCNJ11 (KIR6.2), SLC2A2 (GLUT2), HNF4A (HNF4α), and INS (insulin)—significantly altered disease risk, and in three genes, the risk allele, haplotype, or both had a biologically consistent effect on a relevant physiological trait in the QT study. We examined 35 genes predicted to have their major influence on insulin action, and three (9%)—INSR, PIK3R1, and SOS1—showed significant associations with diabetes. These results confirm the genetic complexity of Type 2 diabetes and provide evidence that common variants in genes influencing pancreatic β-cell function may make a significant contribution to the inherited component of this disease. This study additionally demonstrates that the systematic examination of panels of biological candidate genes in large, well-characterised populations can be an effective complement to positional cloning approaches. The absence of large single-gene effects and the detection of multiple small effects accentuate the need for the study of larger populations in order to reliably identify the size of effect we now expect for complex diseases.     

Linkage relationships of 19 enzyme Loci in maize

Linkage relationships of 19 enzyme loci have been examined. The chromosomal locations of eight of these loci are formally reported for the first time in this paper. These localizations should assist in the construction of additional useful chromosome marker stocks, especially since several of these enzyme loci lie in regions that were previously poorly mapped. Six loci are on the long arm of chromosome 1. The arrangement is (centromere)—Mdh4-mmm-Pgm1-Adh1-Phi-Gdh1, with about 46% recombination between Mdh4 and Gdh1.—Linkage studies with a2 and pr have resulted in the localization of four enzyme genes to chromosome 5 with arrangement Pgm2-Mdh5-Got3-a2-(centromere)-pr-Got2. Pgm2 lies approximately 35 map units distal to a2 in a previously unmapped region of the short arm of 5, beyond ameiotic.—Approximately 23% recombination was observed between Mdh4 and Pgm1 on chromosome 1, while 17% recombination occurred between Mdh5 and Pgm2 on chromosome 5. Similarly, linkages between Idh1 and Mdh1, about 22 map units apart on chromosome 8, and between Mdh2 and Idh2, less than 5 map units apart on chromosome 6, were observed. Thus, segments of chromosomes 1 and 5 and segments of 6 and 8 may represent duplications on nonhomologous chromosomes.     

Rare-Cell Fusion Events between Diploid and Haploid Strains of the Sexually Agglutinative Yeast HANSENULA WINGEI

Diploids of the yeast Hansenula wingei are nonagglutinative and do not form zygotes in mixed cultures with either sexually agglutinative haploid mating type. However, a low frequency of diploid x haploid cell fusions (about 10^-3) is detectable by prototrophic selection. This frequency of rare diploid x haploid matings is not increased after the diploid culture is induced for sexual agglutination. Therefore, we conclude that genes that repress mating are different from those that repress sexual agglutination.——Six prototrophs isolated from one diploid x haploid cross had an average DNA value (µg DNA per 10⁸ cells) of 6.19, compared to 2.53 and 4.35 for the haploid and diploid strains, respectively. Four prototrophs were clearly cell-fusion products because they contained genes from both the diploid and the haploid partners. However, genetic analysis of the prototrophs yielded results inconsistent with triploid meiosis; all six isolates yielded a 2:2 segregation for the mating-type alleles and linked genes.——Mitotic segregation of monosomic (2n-1) cells lacking one homolog of the chromosome carrying the mating-type locus is proposed to explain the rare production of sexually active cells in the diploid cultures. Fusion between such monosomic cells and normal haploids is thought to have produced 3n-1 cells, disomic for the chromosome carrying the mating-type locus. We conclude that in the diploid strain we studied, the physiological mechanisms repressing sexual agglutination and conjugation function efficiently, but events occuring during mitosis lead to a low frequency of genetically altered cells in the population.     

Primary trisomics of rice: origin, morphology, cytology and use in linkage mapping

Twelve primary trisomics of Oryza sativa L. were isolated from the progenies of spontaneous triploids and were transferred by backcrossing to the genetic background of IR36, a widely grown high yielding rice variety. Eleven trisomics can be identified morphologically from one another and from diploids. However, triplo 11 is difficult to distinguish from diploid sibs.—The extra chromosome of each trisomic was identified cytologically at pachytene stage of meiosis, and the chromosomes were numbered according to their length at this stage. The major distinguishing features of each pachytene chromosome were redescribed.—The female transmission rates varied from 15.5% for triplo 1, the longest chromosome, to 43.9% for triplo 12, the shortest chromosome. Seven of the 12 primary trisomics transmitted the extra chromosome through the male. The low level of chromosomal imbalance tolerated by rice and other evidence are interpreted to indicate that this species is a basic diploid.—Genetic segregation for 22 marker genes in the trisomic progenies was studied. Of a possible 264 combinations, involving 22 genes and 12 trisomics, 120 were examined. Marker genes for each of the 12 chromosomes were identified. The results helped establish associations between linkage groups and cytologically identifiable chromosomes of rice for the first time. Relationships between various systems of numbering chromosomes, trisomics, linkage groups and marker genes are described, and a revised linkage map of rice is presented.     

Meiosis in Male DROSOPHILA MELANOGASTER. II. Nonrandom Segregation of Compound-Second Chromosomes

The segregation of compound-second chromosomes in males from two different stocks has been examined. Segregation is random in males from the C(2L)RM4, dp; C(2R)RM4, px stock. Gametes containing only one of the two compound chromosomes comprise 50% of the gametes, and gametes containing either both elements or neither element make up the other 50% of the gametes.——In males from the C(2L)RM, b; C(2R)RM, cn stock, gametes containing either C(2L)RM, b or C(2R)RM, cn make up the majority of the gametes. Gametes containing both chromosomes or neither chromosome account for only 2-3% of the gametes. The nonrandom segregation is due to the C(2R)RM, cn chromosome.——Viability is reduced in flies carrying the C(2R)RM, cn chromosome. This includes larval lethality, delayed development and premature adult lethality. Cytologically, this chromosome contains a large duplication of 2L material, which includes material proximal to region 38 or 39. It is suggested that the viability and segregational properties associated with this chromosome are due to the duplicated 2L material.     

Contribution of Baicalin on the Plasma Protein Binding Displacement and CYP3A Activity Inhibition to the Pharmacokinetic Changes of Nifedipine in Rats In Vivo and In Vitro

Baicalin purified from the root of Radix scutellariae is widely used in clinical practices. This study aimed to evaluate the effect of baicalin on the pharmacokinetics of nifedipine, a CYP3A probe substrate, in rats in vivo and in vitro. In a randomised, three-period crossover study, significant changes in the pharmacokinetics of nifedipine (2 mg/kg) were observed after treatment with a low (0.225 g/kg) or high (0.45 g/kg) dose of baicalin in rats. In the low- and high-dose groups of baicalin-treated rats, C _max of total nifedipine decreased by 40%±14% (P<0.01) and 65%±14% (P<0.01), AUC_0–∞ decreased by 41%±8% (P<0.01) and 63%±7% (P<0.01), V_d increased by 85%±43% (P<0.01) and 224%±231% (P<0.01), and CL increased by 97%±78% (P<0.01) and 242%±135% (P<0.01), respectively. Plasma protein binding experiments in vivo showed that C _max of unbound nifedipine significantly increased by 25%±19% (P<0.01) and 44%±29% (P<0.01), respectively, and there was a good correlation between the unbound nifedipine (%) and baicalin concentrations (P<0.01). Furthermore, in vitro results revealed that baicalin was a competitive displacer of nifedipine from plasma proteins. In vitro incubation experiments demonstrated that baicalin could also competitively inhibit CYP3A activity in rat liver microsomes in a concentration-dependent manner. In conclusion, the pharmacokinetic changes of nifedipine may be modulated by the inhibitory effects of baicalin on plasma protein binding and CYP3A–mediated metabolism.     

A Genetic Polymorphism in Coumarin 7-Hydroxylation: Sequence of the Human CYP2A Gnes and Identification of Variant CYP2A6 Alleles

A group of human cytochrome P450 genes encompassing the CYP2A, CYP2B, and CYP2F subfamilies were cloned and assembled into a 350-kb contig localized on the long arm of chromosome 19. Three complete CYP2A genes—CYP2A6, CYP2A7, and CYP2A13—plus two pseudogenes truncated after exon 5, were identified and sequenced. A variant CYP2A6 allele that differed from the corresponding CYP2A6 and CYP2A7 cDNAs previously sequenced was found and was designated CYP2A6ν2. Sequence differences in the CYP2A6ν2 gene are restricted to regions encompassing exons 3, 6, and 8, which bear sequence relatedness with the corresponding exons of the CYP2A7 gene, located downstream and centromeric of CYP2A6ν2, suggesting recent gene-conversion events. The sequencing of all the CYP2A genes allowed the design of a PCR diagnostic test for the normal CYP2A6 allele, the CYP2A6ν2 allele, and a variant—designated CYP2A6ν1—that encodes an enzyme with a single inactivating amino acid change. These variant alleles were found in individuals who were deficient in their ability to metabolize the CYP2A6 probe drug coumarin. The allelic frequencies of CYP2A6ν1 and CYP2A6ν2 differed significantly between Caucasian, Asian, and African-American populations. These studies establish the existence of a new cytochrome P450 genetic polymorphism.     

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.     

Heteromorphism for Heterokaryon Incompatibility Genes in Natural Populations of NEUROSPORA CRASSA

Five Neurospora crassa isolates from each of three sites in Louisiana were compared for genotype at five heterokaryon incompatibility (het) loci. The comparisons were made using duplications (partial diploids), based on the fact that duplications heterozygous for het loci have strikingly abnormal phenotypes which greatly facilitate the study of such genes. Duplications were synthesized in crosses between the wild strains (normal chromosome sequence) and testers of defined het genotype and having duplication-producing chromosome rearrangements. Crosses segregating for phenotypes characteristic of duplications heterozygous for het loci indicated allelic differences between testers and wild strains for specific het genes. Whenever a wild strain differed from a tester for a specific het locus, but another wild strain did not, the two wild strains could be inferred to differ from each other.—No two isolates from any site were heterokaryon compatible (of identical het genotype), despite the fact that all isolates from each of two sites occurred within several meters of each other. Heteromorphism was found for all five genes studied at one site, four genes at another site, and three at another. Intra- and interpopulation differences between strains were approximately the same.—Confirmation is also provided that two het genes originally detected in duplications are in fact heterokaryon incompatibility loci.     

Location of the LSP-1 Genes in Drosophila Species by IN SITU Hybridization

The locations of the larval serum protein one (LSP-1) α, β and γ genes were determined in Drosophila melanogaster and in 14 other species of Drosophila by in situ hybridization to polytene chromosomes. The LSP-1 α gene mapped to bands 11B on the X chromosome, the LSP-1 β gene mapped to bands 21D-E on chromosome 2L, and the LSP-1 γ gene mapped to band 61A in all the melanogaster subgroup species. In eight other species, both the LSP-1 α and β genes mapped to one site on Muller's element E which corresponds to chromosome 3R of D. melanogaster. No hybridization of LSP-1 γ was detected in these eight species. Restriction enzyme digestion and analysis of genomic DNA by filter transfer hybridization confirmed the presence of LSP-1 α-like and β-like genes in seven of these species. These results are discussed with respect to conservation of the chromosomal elements in the genus Drosophila.     

Hemoglobin Constant Spring among Southeast Asian Populations: Haplotypic Heterogeneities and Phylogenetic Analysis

Background

Hemoglobin Constant Spring (Hb CS) is an abnormal Hb caused by a mutation at the termination codon of α2-globin gene found commonly among Southeast Asian and Chinese people. Association of Hb CS with α°-thalassemia leads to a thalassemia intermedia syndrome commonly encountered in the region. We report chromosome background and addressed genetic origins of Hb CS observed in a large cohort of Hb CS among Southeast Asian populations.

Materials and Methods

A study was done on 102 Vietnamese (aged 15–49 year-old) and 40 Laotian (aged 18–39 year-old) subjects with Hb CS and results compared with 120 Hb CS genes in Thailand. Hematological parameters were recorded and Hb analysis was performed using capillary electrophoresis. Hb CS mutation and thalassemia genotypes were defined by DNA analysis. Six DNA polymorphisms within α-globin gene cluster including 5’Xba I, Bgl I, Inter-zeta HVR, AccI, RsaI and αPstI 3’, were determined using PCR-RFLP assay.

Results

Nine different genotypes of Hb CS were observed. In contrast to the Thai Hb CS alleles which are mostly linked to haplotype (+—S + + -), most of the Vietnamese and the Laotian Hb CS genes were associated with haplotype (+—M + + -), both of which are different from that of the European Hb CS.

Conclusions

Hb CS is commonly found in combination with other thalassemias among Southeast Asian populations. Accurate genotyping of the cases requires both hematologic and DNA analyses. At least two independent origins are associated with the Hb CS gene which could indirectly explain the high prevalence of this Hb variant in the region.     

DNA methylome profiling identifies novel methylated genes in African American patients with colorectal neoplasia

The identification of genes that are differentially methylated in colorectal cancer (CRC) has potential value for both diagnostic and therapeutic interventions specifically in high-risk populations such as African Americans (AAs). However, DNA methylation patterns in CRC, especially in AAs, have not been systematically explored and remain poorly understood. Here, we performed DNA methylome profiling to identify the methylation status of CpG islands within candidate genes involved in critical pathways important in the initiation and development of CRC. We used reduced representation bisulfite sequencing (RRBS) in colorectal cancer and adenoma tissues that were compared with DNA methylome from a healthy AA subject’s colon tissue and peripheral blood DNA. The identified methylation markers were validated in fresh frozen CRC tissues and corresponding normal tissues from AA patients diagnosed with CRC at Howard University Hospital. We identified and validated the methylation status of 355 CpG sites located within 16 gene promoter regions associated with CpG islands. Fifty CpG sites located within CpG islands—in genes ATXN7L1 (2), BMP3 (7), EID3 (15), GAS7 (1), GPR75 (24), and TNFAIP2 (1)—were significantly hypermethylated in tumor vs. normal tissues (P < 0.05). The methylation status of BMP3, EID3, GAS7, and GPR75 was confirmed in an independent, validation cohort. Ingenuity pathway analysis mapped three of these markers (GAS7, BMP3 and GPR) in the insulin and TGF-β1 network—the two key pathways in CRC. In addition to hypermethylated genes, our analysis also revealed that LINE-1 repeat elements were progressively hypomethylated in the normal-adenoma-cancer sequence. We conclude that DNA methylome profiling based on RRBS is an effective method for screening aberrantly methylated genes in CRC. While previous studies focused on the limited identification of hypermethylated genes, ours is the first study to systematically and comprehensively identify novel hypermethylated genes, as well as hypomethylated LINE-1 sequences, which may serve as potential biomarkers for CRC in African Americans. Our discovered biomarkers were intimately linked to the insulin/TGF-B1 pathway, further strengthening the association of diabetic disorders with colon oncogenic transformation.     

Gene-Centromere Mapping in Rainbow Trout: High Interference over Long Map Distances

Ten enzyme loci were mapped in relation to their centromeres in gynogenetic diploid rainbow trout. Gene-centromere map distances, calculated under the assumption of complete interference, range from 1.1 cM for Ldh4 to 50 cM for Sod1. The Idh2 and Est1 loci are linked on the same chromosome arm.—The observation of close to 100% heterozygous gynogenetic diploids for the Sod1 and Mdh3,4 loci suggests that near-complete interference occurs on the chromosome arms carrying these loci. The high interference observed in this study and in several other species of fish may be related to the small size of fish chromosome arms.—Comparisons of map locations for the Ldh3 and Ldh4 and the Mdh3 and Mdh4 loci, which were duplicated by a tetraploid event in the evolution of salmonid fish, reveal that they are located at similar distances from their centromeres. Comparative mapping of loci duplicated longer ago shows more variation in map location.—The high proportion of heterozygotes for some loci after gynogenesis involving second polar body retention demonstrates that this is not a practical method for producing homozygous inbred lines in rainbow trout; treatments suppressing the first cell division are more promising for this purpose.