QTL mapping of temperature sensitivity reveals candidate genes for thermal adaptation and growth morphology in the plant pathogenic fungus Zymoseptoria tritici.

Different thermal environments impose strong, differential selection on populations, leading to local adaptation, but the genetic basis of thermal adaptation is poorly understood. We used quantitative trait locus (QTL) mapping in the fungal wheat pathogen Zymoseptoria tritici to study the genetic architecture of thermal adaptation and identify candidate genes. Four wild-type strains originating from the same thermal environment were crossed to generate two mapping populations with 263 (cross 1) and 261 (cross 2) progeny. Restriction site-associated DNA sequencing was used to genotype 9745 (cross 1) and 7333 (cross 2) single-nucleotide polymorphism markers segregating within the mapping population. Temperature sensitivity was assessed using digital image analysis of colonies growing at two different temperatures. We identified four QTLs for temperature sensitivity, with unique QTLs found in each cross. One QTL had a logarithm of odds score >11 and contained only six candidate genes, including PBS2, encoding a mitogen-activated protein kinase kinase associated with low temperature tolerance in Saccharomyces cerevisiae. This and other QTLs showed evidence for pleiotropy among growth rate, melanization and growth morphology, suggesting that many traits can be correlated with thermal adaptation in fungi. Higher temperatures were highly correlated with a shift to filamentous growth among the progeny in both crosses. We show that thermal adaptation has a complex genetic architecture, with natural populations of Z. tritici harboring significant genetic variation for this trait. We conclude that Z. tritici populations have the potential to adapt rapidly to climate change and expand into new climatic zones.

The effect of a leptin single nucleotide polymorphism on quality grade, yield grade, and carcass weight of beef cattle.

Feedlot producers could optimize the value of cattle in a given market grid if they were able to improve the uniformity of the body composition between cattle among loads. Allelic variation due to a single nucleotide transition (cytosine [C] to thymine [T] transition that results in a Arg25Cys) has been demonstrated to be associated with higher leptin mRNA levels in adipose tissue and increased fat deposition in mature beef, but the effect on economically important carcass traits has not been investigated in either market-ready steers or heifers. Therefore, the objective of this study was to determine the effects of a leptin SNP on the quality grade (QG), yield grade (YG), and weight of beef carcasses. A slaughter trial was conducted using 1,435 crossbred finished heifers and 142 crossbred finished steers as they entered the slaughter facility. Canada QG tended (main effect of genotype P = 0.16, but P < 0.01 for both CC vs. TT and CT vs. TT) to be affected by leptin genotype. Specifically, 7.6 and 7.1% more TT carcasses graded Canada AAA or higher than the CT and CC carcasses, respectively, which supports the suggestion that the leptin SNP is associated with carcass fat. The proportion of carcasses grading Canada YG 1, 2, or 3 was affected (P < 0.01, P = 0.05, and P = 0.02 for YG 1, 2, and 3) by leptin genotype. The proportion of TT carcasses of Canada YG 1 was 12.5 and 15.1% lower than that of CT and CC carcasses, respectively, indicating that rearing animals under the same management and feeding system may result in greater carcass fat and a lower probability of the proportion of carcasses grading YG 1 within certain genotypes. The carcass weights of animals with the CC genotype tended (P = 0.07) to be higher than those of the TT genotype (365.5 vs. 362.3 kg). No significant difference was observed between the TT and CT genotypes in carcass weight. The observed associations between leptin genotype and carcass characteristics may represent an opportunity to genetically identify animals that are most likely to reach specific marketing groups.

First Report of Phaeomoniella chlamydospora on Vitis vinifera, Vitis labrusca, and French American Hybrids in Pennsylvania and New York.

Phaeomoniella chlamydospora (W. Gams, Crous, M.J. Wingfield. & L. Mugnai) Crous & Gams (= Phaeoacremonium chlamydosporum) was isolated during the growing seasons of 2001 and 2002 from roots, trunks, and cordons of grapevines including cultivars Concord, Niagara, Steuben, Catawba, Dutchess, DeChaunac, Vidal, Seyval, Chambourcin, Chardonnay, Riesling, Sangiovese, Dolcetto, Baco Noir, Merlot, Villard, Pinot Gris, GR7, and 3309C root stock representing 18 locations in Eastern, Central, and Lake Erie regions of Pennsylvania as well as the Lake Erie and Finger Lakes regions of New York. P. chlamydospora was isolated from 89% of samples from vines 3 to 45 years old showing decline symptoms in the field. Isolates were identified based on a previous description (1) and by internal transcribed spacer (ITS1-5.8S-ITS2) rDNA sequences identical to those of P. chlamydospora isolated from Vitis vinifera from Italy (ex-type culture CBS229.95, GenBank Accession No. AF197973). P. chlamydospora is firmly established as a member of the petri and esca disease complex and as a pathogen of grapevines (2,3). To test pathogenicity of our isolates, approximately 30 µl of a 106 conidia/ml suspension, obtained from six isolates, was injected into the pith of 60 single-node, dormant, unrooted cuttings of '3309C' and 'Concord'. Ten control cuttings of 'Concord' and '3309C' were injected with an equal volume of sterile distilled water. From 24 to 32 weeks after inoculations, all P. chlamydospora-inoculated cuttings exhibited dark streaking of the vascular tissue extending 45 to 50 mm from the point of inoculation. The vascular streaking observed in inoculated plants was identical to symptoms observed in declining vines in the vineyard. Vascular streaking was absent in the controls. P. chlamydospora was isolated as a monoculture from regions of vascular streaking in 89% of inoculated cuttings. P. chlamydospora was not isolated from the water-treated controls. P. chlamydospora is widespread and readily isolated from declining grapevines in Pennsylvania, New York, and other national and international grape growing regions. To our knowledge, this is the first report of P. chlamydospora from the cultivars cited above in Pennsylvania and New York. References: (1) M. Groenewald et al. Mycol. Res. 105:651, 2001. (2) Phytopathol. Mediterr. 39(1), 2000. (3) Phytopathol. Mediterr. 40, Supplement 2001.

2-Amino-4,6-diarylpyridines as novel ligands for the estrogen receptor.

We have prepared a novel series of 2-amino-4,6-diarylpyridines that function as ligands of estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta). These compounds bind to both ERalpha and ERbeta with a modest selectivity for the alpha subtype. The most potent of these analogues, compound 19, has a K(i)=20nM at ERalpha. These molecules represent a novel template for designing potentially useful ligands for the estrogen receptor.

Molecular Mechanics (MM3) Calculations on Oxygen-Containing Phosphorus (Coordination IV) Compounds.

An MM3 force field has been developed for small oxygen-containing phosphorus (coordination IV) compounds. Experimental electron and neutron diffraction, X-ray, and infrared spectral data were utilized in parametrization when available. Results from previous ab initio calculations at both the restricted Hartree-Fock (RHF) and second-order Møller-Plesset (MP2) levels of theory with the standard 6-31G basis set supplemented missing structural data, rotational profiles, and vibrational frequencies. Negative hyperconjugation (the anomeric effect) affects the structures and energies of the molecules under study which contain one or more sp(3)-hybridized oxygen atoms. Natural bond order (NBO) analysis was helpful in identifying the key orbital interactions involved in this effect.

Molecular modeling studies of some choline acetyltransferase inhibitors.

Choline acetyltransferase (ChAT) inhibitors related to trans-1-methyl-4- (1-naphthylvinyl)-pyridinium (NVP+) have been assumed to depend upon a nearly or completely planar conformation for their enzyme-inhibitor interaction. In an effort to investigate the geometries and preferred conformations for these compounds, geometry optimizations using the semiempirical molecular orbital method AM1 and a modified version of the molecular mechanics method MM2(92) have been carried out. The results indicate that the active inhibitors are either planar or nearly coplanar, lying in relatively flat potential wells in the vicinity of the corresponding planar structures. When nonplanarity is favored, one ring is twisted out of the plane by approximately 30 degrees. Where steric features significantly deter assumption of a nearly planar conformation, the analogs are inactive. The inactivity of analogs bearing tricyclic aryl groups appears to result from bulk-related hindrance to ChAT receptor binding rather than lack of coplanarity.

Computer-assisted molecular modeling: indispensable tools for molecular pharmacology.

Ball-and-stick mechanical models, typically associated with chemists, have been helpful in understanding structural problems and the relationship between structure and biologic activity. With progress in computer speed, graphics performance, and software innovation, molecules of biological interest can be subjected to rigorous calculations. Computational chemistry and biology are rooted in the belief that theoretical physics can be used to calculate accurate molecular structures. Although in its infancy, computer-assisted molecular modeling is gaining attention and acceptability as an increasing number of researchers turn their attention toward rational molecular design. The trend to use theoretical methods can be traced to the greater availability of computer graphics work-stations, decreasing computer costs, faster central processing units, more robust algorithms, and "user-friendly" software codes. Every major pharmaceutical company has invested in these resources to reduce the time it takes to design and develop pharmaceutical agents. Because of the vast financial and manpower investments needed to introduce a single drug, medicinal chemists and pharmacologists are interested in understanding and predicting drug action at the molecular level. Although drug action is still poorly understood, molecular modeling should reduce some of the labor in the development of pharmaceutical agents.

Molecular taxonomic relationships in the genus Vigna based on RFLP analysis.

The taxonomy of the genus Vigna has been primarily based on morphological attributes. We have used 27 genomic clones from soybean, common bean, mungbean and cowpea to examine restriction fragment length polymorphism (RFLP) among 44 accessions of different species belonging to four subgenera of the genus Vigna. One accession each of common bean (Phaseolus vulgaris) and soybean (Glycine max) was included in the study. Total DNA from the various genotypes was digested with one restriction enzyme (Eco RV). Results of a numerical taxonomic analysis showed a high level of genetic variation within the genus with a remarkably higher amount of variation associated with Vigna sp. from Africa relative to those from Asia. The distinctness of the Asiatic grams in subgenus Ceratotropis, cowpea in section Catiang, bambara groundnut (V. subterranean) and members of the subgenus Plectotropis was elucidated by this study. Members of the subgenus Plectotropis were closer in genome homology to those of subgenus Vigna section Catiang than to those of subgenus Ceratotropis. The relative positions of some genotypes to one another on the dendrogram and minimum spanning tree were discussed in regard to hybridisations aimed generating well-saturated genomic maps and interspecies transfer of desirable genes.

Chromosomes and other prognostic factors in acute lymphoblastic leukaemia: a long-term follow-up.

Cytogenetic, clinical and laboratory features at diagnosis were examined in a group of 80 children with acute lymphoblastic leukaemia (ALL) who had been followed up for a minimum of 5 1/2 years. The 17 (21%) with high hyperdiploidy tended to have low leucocyte counts and common ALL, but their favourable outcome (75% event-free survival) was independent of these factors. No patient with hypodiploidy survives while the pseudodiploid and normal groups have an intermediate prognosis. Cytogenetic analysis showed examples of patients with the well-recognized translocations and a number with apparently unique ones. Among the latter were some long-term survivors. We conclude that cytogenetic analysis identifies a good risk group of patients who remain well on long-term follow-up, but that the presence of a translocation does not necessarily imply a poor outcome.

Detection of the chromosomally abnormal clone in acute lymphoblastic leukemia.

Bone marrow and peripheral blood from 121 children with acute lymphoblastic leukemia spent 3 hours in transit before being cultured. Cultures were harvested directly, after 24, 48, or 72 hours, and following methotrexate synchronization. Details of techniques are given. Analyzable mitoses were obtained in 78 cases. Failures contained no detectable clone and fewer than five mitoses (27 cases) or fewer than five analyzable mitoses (16 cases). A clone was found in 44 cases. In some cases the clone was found in two or more cultures, conversely a clone in the direct harvest was not always found after overnight culture and vice versa. Hyperdiploid clones were always found in the bone marrow and frequently in two or more cultures. Pseudodiploid clones found in the blood in four cases evaded detection in the bone marrow. The use of methotrexate did not yield prometaphase chromosomes or improve the yield of analyzable cells. The number of cultures employed influenced the yield of metaphases and appeared to influence clone detection. More cultures were needed to detect a pseudodiploid clone than to detect a hyperdiploid clone. Detection of a clone may be maximized by using the following combination of cultures: bone marrow direct and overnight, and peripheral blood overnight.

The (4;11) translocation in acute leukaemia of childhood: the importance of additional chromosomal aberrations.

Case reports of four girls and one boy with acute lymphoblastic leukaemia (ALL) or acute myeloid leukaemia (AML) and t(4;11) are presented. The incidence of t(4;11) ascertained at diagnosis in ALL was 2.6% and in AML 5.3%. Four of the children were under 2 years and one was 11 years at diagnosis. Leucocyte counts above 71 X 10(9)/l and liver, spleen and node enlargement were found in all cases. Blasts of the four cases tested at diagnosis were negative to the c-ALL antigen and either TdT+ (ALL) or TdT- (AML M1). Maximum survival was less than 8 months. Additional chromosomal change was found at diagnosis in two cases and in relapse in a third. In the case of AML t(4;11) (q21;p15) was present as a second translocation. Additional numerical changes, in these and other reported cases, included + 6, commonly found in ALL, +8, +19, more often reported in AML. It is suggested that additional chromosomal changes in these cases support cytochemical and surface marker evidence that t(4;11) has a pluripotent target cell, similar to that of the Philadelphia translocation.

Acute lymphoblastic leukaemia with t(4;11) follows neuroblastoma: a late effect of treatment?

The case presented is of a 7-year-old girl who developed acute lymphoblastic leukaemia (ALL) with t(4;11)(q21;q23) 5 years after the onset of neuroblastoma and 4 months after completing treatment for a first relapse. Consideration is given to the relative importance in this case of genetic factors and chemotherapeutic drugs in the etiology of ALL.

Formation of 3-(2'-deoxyribofuranosyl) and 9-(2'-deoxyribofuranosyl) nucleosides of 8-substituted purines by nucleoside deoxyribosyltransferase.

Earlier results suggested that although the N-deoxyribosyltransferase from lactobacilli is a convenient tool for the preparation of analogs of 2'-deoxyadenosine, 8-substituted purines do not act as substrates. However, eight of nine 8-substituted purines that were examined proved to be substrates for the transferase from Lactobacillus leichmannii, and deoxyribonucleosides of four of these bases have been prepared. The substituents at C-8 of the purine greatly affect the rate of deoxyribosyl transfer to the base, and in all cases the rate is slower than transfer to purines lacking an 8-substituent. The 8-substituent also affects the nature of the nucleoside formed. With the electron-donating methyl group at position 8 of adenine, the transferase forms the expected 8-methyl-9-(2'-deoxyribofuranosyl)adenine. However, when purines bearing an electron-withdrawing substituent at the 8-position are used as substrates, the deoxyribosyl moiety is preferentially transferred to N-3 of the base. In the case of 8-trifluoromethyladenine the 3-deoxyribonucleoside is the only product detectable. With 8-bromo or 8-chloroadenine as substrate the 3- and 9-deoxyribonucleosides can both be isolated from the enzymatic reaction mixture. Time course studies indicated that with thymidine and 8-bromoadenine as substrates the 3-deoxyribonucleoside is initially the major product, but that the 9-deoxyribonucleoside becomes the major product after long incubation periods. Negligible interconversion of these nucleosides occurs in the absence of transferase, but conversion in either direction occurs readily in the presence of the enzyme. Significant hydrolysis of pyrimidine and purine deoxyribonucleosides occurs in the presence of the transferase. This was more obvious during the course of reactions involving 8-substituted purines because the slowness of deoxyribosyl transfer required longer incubation periods and larger amounts of enzyme. The hydrolysis is proportional to enzyme concentration, little affected by the nature of the base and is attributed to hydrolysis of a deoxyribosyl derivative of the transferase which is an obligatory intermediate of deoxyribosyl transfer. 8-Trifluoromethyl-3-(2'-deoxyribofuranosyl)adenine, 8-methyl-9-(2'-deoxyribofuranosyl)adenine, and 8-bromo-9-(2'-deoxyribofuranosyl)adenine were tested for their ability to inhibit the growth of CCRF-CEM cells in culture. Unlike the potent 2-halogeno-2'-deoxyadenosine derivatives, these three nucleosides cause less than 50% inhibition at concentrations up to 100 microM.