The molecular genetic differentiation of cultured Saccharomyces strains

A comparative molecular genetic study of cultured Saccharomyces strains isolated from the surface of berries and various fermentation processes showed that baker's yeast and black-currant isolates contain not only Saccharomyces cerevisiae but also S. cerevisiae and S. bayanus var. uvarum hybrids. The molecular karyotyping of baker's, brewer's, and wine yeasts showed their polyploidy. The restriction enzyme analysis of noncoding rDNA regions (5.8S-ITS and IGS2) makes it possible to differentiate species of the genus Saccharomyces and to identify interspecies hybrids. The microsatellite primer (GTG)5 can be used to study the populations of cultured S. cerevisiae strains.     

Identification and molecular mapping of a Fusarium wilt resistant gene in upland cotton

Fusarium wilt (FW) is one of the most economically damaging cotton diseases worldwide, causing yellowing, wilting, defoliation, vascular tissue damage and ultimately death. Identification of molecular markers linked to FW genes is vital to incorporate resistance into elite cotton cultivars. An intraspecific F₂ in Gossypium hirsutum L. was developed by crossing with a highly resistant cultivar Zhongmiansuo 35 (ZMS35) and a susceptible cultivar Junmian 1 to screen simple sequence repeats (SSRs) closely linked to the FW resistance gene. FW was identified in F_2:3 families by evaluating seedling leaf symptoms and vascular tissue damage at plant maturity under natural field infection conditions over 2 years. The results showed that FW resistance segregated in a 3:1 ratio as a simple monogenic trait in F_2:3 families. Molecular mapping identified a FW resistance gene closely linked with the SSR marker JESPR304₋₂₈₀ in chromosome D3(c17). We proposed to name this gene FW ^R . A composite interval mapping method detected four QTLs for FW resistance in Chr.A7(c7), D1(c15), D9(c23) and D3, respectively. Among them, one major QTL (LOD > 20) was tagged near marker JESPR304 within an interval of 0.06–0.2 cM, and explained over 52.5–60.9% of the total phenotypic variance. The data confirmed the existence of a major gene in Chr.D3. This is the first report of molecular mapping of a major gene contributing FW resistance in cotton. The present research therefore provides an opportunity to understand the genetic control of resistance to FW and conduct molecular marker-assisted selection breeding to develop FW resistant cultivars.     

Purification and partial characterisation of pectin methylesterase produced by Fusarium asiaticum

Fusarium asiaticum is the predominant causal agent of Fusarium head blight (FHB) in China. When grown in liquid cultures containing potato tuber extract as the sole carbon source, F. asiaticum (strain 7071) from wheat (China) produced pectin methylesterase (PME), polygalacturonase (PG), and pectin lyase (PNL). The activity of these pectolytic enzymes was detected by a gel diffusion assay. Three forms of PME were identified in a culture filtrate of F. asiaticum. Two forms of PME with molecular weights of 31 kDa and 42.5 kDa, as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), were purified using a combination of chromatographic techniques. These PMEs did not bind to Concanavalin A (Con A), which was confirmed by rechromatography using a Con A agarose column. The 31 kDa purified PME was thermostable in a temperature range of 25-55 °C. The optimal pH for the PME of F. asiaticum was 6.5. This research provides the basis for future investigations of pectolytic enzymes from F. asiaticum.     

Regulation of metabolic products and gene expression in Fusarium asiaticum by agmatine addition

The metabolic products resulting from the cultivation of F. asiaticum in agmatine were identified using capillary electrophoresis–time of flight mass spectrometry. Glyoxylic acid was detected from fungal cultures grown in agmatine, while it was absent in control cells. The abundance of other metabolic products of the glycolytic pathway also increased because of agmatine; however, there was no increase in the amounts of pyruvic acid or metabolites from the tricarboxylic acid cycle. Moreover, gene expression levels within Fusarium asiaticum exposed to agmatine were analyzed by DNA microarray. Changes in gene expression levels directed the changes in metabolic products. Our results suggest that acetyl coenzyme A, which is a starting substrate for the biosynthesis of deoxynivalenol (DON), was simultaneously produced by activated β-oxidation. Furthermore, the content of 4-aminobutyrate (GABA) was increased in the agmatine addition culture medium. GABA can be synthesized from agmatine through putrescine and might influence the regulation of DON-related genes.     

Current status of preimplantation genetic diagnosis in Japan

This is a retrospective study aimingto clarify the current status of preimplantation genetic diagnosis (PGD) in Japan. Our data were collected from 12 facilities between September 2004 and September 2012, and entered into a database. A majority of PGD in Japan was performed for balanced structural chromosomal abnormalities in couples with recurrent miscarriage. PGD for monogenic diseases was performed only in two facilities. The average maternal age was 38 years for monogenic diseases and 40 years for chromosomal abnormalities. Overall there have been671 cycles to oocyte retrieval reported. Of these cycles, 85% (572 cycles)were for chromosomal abnormalities, and 15% (99 cycles) for monogenic diseases. Diagnosis rates in the current study were 70.8% for monogenic diseases and 94.0% for chromosomal abnormalities. Rates of embryo transfer of PGD were 62.7% for monogenic diseases and 25.5% for chromosomal abnormalities. Clinical pregnancy rates per embryo transfer were 12.0% for monogenic diseases and 35.6% for chromosomal abnormalities. Our study is the first PGD report from all facilities which had the approval of the ethics committee of the Japanese Society of Obstetrics and Gynecology. We have built a basis for gathering continuous PGD data in Japan.     

Isolation and genetic analysis of Chlamydomonas reinhardtii strains resistant to cadmium

In Chlamydomonas reinhardtii, cadmium induces reduction of growth, reduction of chlorophyll content, and lethality. The toxicity was higher in a cell wall-deficient strain than in the wild type. By growing the cells on agar medium containing cadmium at concentrations inducing high lethality, stable resistant clones were isolated. The resistance was due to a nuclear mutation (cadAR) which probably preexisted in the wild-type cell population, as suggested by the fluctuation test. A double mutant (cadAR cadBR) was selected on media containing higher concentrations of cadmium. The cadBR mutation, which is unlinked to cadAR, determines a resistance intermediate between the CadAR mutant and the wild-type strain. Both cadAR and cadBR mutations are partially dominant.     

Identification and Genetic Division of Fusarium graminearum and Fusarium asiaticum by Species‐Specific SCAR Markers

Fusarium head blight (FHB), also called scab, is a devastating and insidious disease of cereals including wheat (Triticum spp.) and barley (Hordeum vulgare L.) worldwide. Apart from direct yield losses, the most serious concern about FHB is the contamination of the crop with mycotoxins, which pose a health risk to human and livestock. Recent research reported that phylogenetic species F. asiaticum (Fa) and F. graminearum (Fg) were the major causal agents of FHB from infected wheat heads in China. To investigate the population structure of Fusarium species in China by species‐specific as well as the chemotype‐specific markers, sequence‐related amplified polymorphism (SRAP) markers were screened on representative isolates of F. asiaticum‐NIV, F. asiaticum‐ 3ADON and F. graminearum‐15ADON to find amplification products characteristic of either species or chemotypes. Selected amplified fragments were cloned and sequenced so that sequence‐characterized amplified region (SCAR) primer pairs could be developed which permit specific detection of Fusarium species using conventional PCR. Primer pairs SCAR‐Fa1 and SCAR‐Fg1 were confirmed to be able to amplify specific products only in F. asiaticum and F. graminearum isolates, respectively. These species‐specific primers were applied to determine genetic division of F. asiaticum and F. graminearum isolates collected in Yangtze–Huaihe valley. The results indicated that F. asiaticum was the predominant species causing FHB in this wheat production area. It is the first report that SRAP markers were adapted for species characterization in Fusarium isolates.     

Isolation and genetic analysis of Chlamydomonas reinhardtii strains resistant to cadmium.

In Chlamydomonas reinhardtii, cadmium induces reduction of growth, reduction of chlorophyll content, and lethality. The toxicity was higher in a cell wall-deficient strain than in the wild type. By growing the cells on agar medium containing cadmium at concentrations inducing high lethality, stable resistant clones were isolated. The resistance was due to a nuclear mutation (cadAR) which probably preexisted in the wild-type cell population, as suggested by the fluctuation test. A double mutant (cadAR cadBR) was selected on media containing higher concentrations of cadmium. The cadBR mutation, which is unlinked to cadAR, determines a resistance intermediate between the CadAR mutant and the wild-type strain. Both cadAR and cadBR mutations are partially dominant.     

Advances in molecular genetic diagnosis of leukemia]

Recently, Molecular genetics has remarkably advanced and it is introduced in medicine. The use of recombinant DNA methods for the diagnosis of leukemias is reported with special reference to the contribution of cytogenetic findings, such as specific chromosome aberrations previously obtained. Therefore, cytogenetic studies on Ph1 chromosome and other specific aberrations found in leukemias are historically reviewed. Using Southern blotting, PFGE, PCR, and in situ chromosome mapping techniques we have analyzed many cases with CML and cases with ALL. We found M-bcr rearrangements not only in standard Ph1, but also in complex types and in Ph1 (-) ve CML. Chromosomal in situ hybridization was very informative identifying transposition of bcr and abl genes between chromosomes 22 and 9. In this connection, FISH (fluorescence in situ hybridization) technique was developed by us, which is expected to have an exceptional power of analysis. ALL had either M-bcr or m-bcr rearrangements, the latter being identified by PFGE. Next, application of PCR technique that enables to obtain more than 10(5) copies of target sequences could monitor minimal residual diseases in CML. Recently, the relevant gene were cloned respectively in FAB-M2 and APL (FAB-M3), so that detection of minimal residual diseases will be successfully performed in these types of leukemia. Finally, targeting chemotherapy using antisense sequences is prospectively described.     

Rett syndrome molecular diagnosis and implications in genetic counseling

Rett syndrome is a rare genetic X-linked dominant disorder. This syndrome is the most frequent cause of mental retardation in girls. In the classical form of the disease, the presenting signs and the course of development are characteristic. However clinical diagnosis can be very difficult when the expression is not in the classical form. Mutations in MeCP2 are responsible for 80% of cases. When MeCP2 mutation is found in an index case, genetic counseling is similar to that in other X-linked dominant genetic diseases. However, mutations in this gene can cause a spectrum of atypical forms. On the other hand, other genetic conditions like translocations, sex chromosome numerical anomalies, and mutations in other genes can complicate genetic counseling in this syndrome. We present the first case of molecular diagnosis of Rett syndrome in Iran and discuss the recent developments in its genetic counseling.     

Expression profile analysis of wild-type and fcc1 mutant strains of Fusarium verticillioides during fumonisin biosynthesis

Fusarium verticillioides produces a group of mycotoxins known as fumonisins that are associated with a variety of mycotoxicoses in humans and animals. In this study, DNA microarrays were constructed with expressed sequence tags (ESTs) from F. verticillioides. To identify genes with patterns of expression similar to the fumonisin biosynthetic (FUM) genes, the microarray was probed with labeled cDNAs originating from a wild-type strain and a fcc1 mutant grown on maize and in a defined medium adjusted to either pH 3 or pH 8. The comparative analyses revealed differential expression of genes corresponding to 116 ESTs when the fungal strains were grown on maize. Under different pH conditions, 166 ESTs were differentially expressed, and 19 ESTs were identified that displayed expression patterns similar to the FUM ESTs. These results provide candidate genes with potential roles in fumonisin biosynthesis.     

Alteration in molecular species of phosphatidylethanolamine between anesthetic resistant and sensitive strains of drosophila melanogaster

An analysis of lipid composition was carried out in resistant and sensitive strains of $\text{Drosophila}$$\text{melanogaster}$. Amount of total lipid and amount of phosphate of phospholipids were not different from each other in both strains. Phosphatidylethanolamine (PE) and phosphatidylcholine (PC) did not differ in amount between both strains. Determination of molecular species of PE using gas chromatography (GC) and GC-mass spectrometry showed that the resistant strain had increased 34:2 and decreased 36:2, 36:3 and 36:5 relative to the sensitive strain. The molecular species of PC did not differ between the two strains. Chromosomal analysis revealed that the alterations in 34:2 and 36:2 of PE were regulated by the X- and third chromosomes of the resistant strain. Therefore, the changes in PE may explain the mechanism of anesthetic resistance because genetic analyses indicate that these chromosomes have more influence on the anesthetic resistant traits of the resistant strain.     

Comparison of biochemical and molecular genetic approaches for identification of environmental strains

In order to confirm the taxonomic position of environmental strains determined based on their biochemical, cultural, and morphological characteristics, molecular genetic identification was carried out. A number of problems in identification of microorganisms were shown to be associated with contamination of the cultures in the course of isolation. Advantages of a comprehensive approach—combining 16S rRNA gene sequencing with a set of biochemical, cultural, and morphological parameters—for identification of microorganisms isolated from environmental objects and clinical samples are discussed.     

Gibberellin biosynthesis and gibberellin oxidase activities in Fusarium sacchari,Fusarium konzum and Fusarium subglutinans strains

Several isolates of three Fusarium species associated with the Gibberella fujikuroi species complex were characterized for their ability to synthesize gibberellins (GAs): Fusarium sacchari (mating population B), Fusarium konzum (mating population I) and Fusarium subglutinans (mating population E). Of these, F. sacchari is phylogenetically related to Fusarium fujikuroi and is grouped in the Asian clade of the complex, while F. konzum and F. subglutinans are only distantly related to Fusarium fujikuroi and belong to the American clade. Variability was found between the different F. sacchari strains tested. Five isolates (B-12756; B-1732, B-7610, B-1721 and B-1797) were active in GA biosynthesis and accumulated GA₃ in the culture fluid (2.76–28.4 μg/mL), while two others (B-3828 and B-1725) were inactive. GA₃ levels in strain B-12756 increased by 2.9 times upon complementation with ggs2 and cps-ks genes from F. fujikuroi. Of six F. konzum isolates tested, three (I-10653; I-11616; I-11893) synthesized GAs, mainly GA₁, at a low level (less than 0.1 μg/mL). Non-producing F. konzum strains contained no GA oxidase activities as found for the two F. subglutinans strains tested. These results indicate that the ability to produce GAs is present in other species of the G. fujikuroi complex beside F. fujikuroi, but might differ significantly in different isolates of the same species.     

A genetic and molecular profile of third chromosome centric heterochromatin in Drosophila melanogaster.

In this review, we combine the results of our published and unpublished work with the published results of other laboratories to provide an updated map of the centromeric heterochromatin of chromosome 3 in Drosophila melanogaster. To date, we can identify more than 20 genes (defined DNA sequences with well-characterized functions and (or) defined genetic complementation groups), including at least 16 essential loci. With the ongoing emergence of data from genetic, cytological, and genome sequencing studies, we anticipate continued, substantial progress towards understanding the function, structure, and evolution of centric heterochromatin.     

Six strains of human immunodeficiency virus type 1 isolated in Japan and their molecular phylogeny

Summary Five strains of human immunodeficiency virus type 1 (HIV-1) were isolated from five Japanese hemophilia patients. Two isolates, HIV1[GUN-1] and HIV-1[GUN-2], were from brother patients with hemophilia B and the other three isolates, HIV-I[GUN-3], HIV-1[GUN-4], and HIV1[GUN-5], were from hemophilia A patients. Another HIV-1 strain, HIV-1[GUN-6], was isolated from a Canadian male homosexual with AIDS. The restriction endonuclease cleavage maps of the proviral genomes of these six HIV-1 strains revealed that they were apparently different from each other. The phylogenetic trees constructed using restriction maps and nucleotide sequences were quite similar, indicating that phylogenetic analyses of Japanese HIV-1 isolates can be done using restriction maps of the proviruses. Phylogenetic analyses showed that they were more closely related to HIV-1s which had been reported to be isolated from homosexual patients in the United States than those isolated from African patients. In particular, GUN-1 and GUN-2 isolates were on the branch of a San Francisco isolate, ARV2, while GUN-5 and GUN-6 isolates were on the branch of HTLV-III_B-related isolates.Offprint requests to: H. Hoshino