Molecular characterization of the afl-1 locus in Aspergillus flavus.

An unusual mutation at the afl-1 locus, affecting aflatoxin biosynthesis in Aspergillus flavus 649, was investigated. The inability of strain 649 to produce aflatoxin was found to be the result of a large (greater than 60 kb) deletion that included a cluster of aflatoxin biosynthesis genes. Diploids formed by parasexual crosses between strain 649 and the aflatoxigenic strain 86 did not produce aflatoxin, indicating the dominant nature of the afl-1 mutation in strain 649. In metabolite feeding experiments, the diploids did not convert three intermediates in the aflatoxin pathway to aflatoxin. Northern (RNA blot) analysis of the diploids grown in medium conducive for aflatoxin production indicated that the aflatoxin pathway genes nor1, ver1, and omt1 were not expressed; however, there was low-level expression of the regulatory gene aflR. Pulsed-field electrophoresis gels indicated a larger (6 Mb) chromosome in strain 649 than the apparently homologous (4.9 Mb) chromosome in strain 86. The larger chromosome in strain 649 suggests that a rearrangement occurred in addition to the deletion. From these data, we proposed that a trans-sensing mechanism in diploids is responsible for the dominant phenotype associated with the afl-1 locus in strain 649. Such a mechanism is known in Drosophila melanogaster but has not been described for fungi.     

Molecular characterization of the mouse agouti locus.

The agouti (a) locus acts within the microenvironment of the hair follicle to regulate coat color pigmentation in the mouse. We have characterized a gene encoding a novel 131 amino acid protein that we propose is the one gene associated with the agouti locus. This gene is normally expressed in a manner consistent with a locus function, and, more importantly, its structure and expression are affected by a number of representative alleles in the agouti dominance hierarchy. In addition, we found that the pleiotropic effects associated with the lethal yellow (Ay) mutation, which include pronounced obesity, diabetes, and the development of neoplasms, are accompanied by deregulated overexpression of the agouti gene in numerous tissues of the adult animal.     

Molecular characterization of the murine argininosuccinate synthetase locus.

The cDNA and gene encoding murine argininosuccinate synthetase were cloned and characterized. The cDNA sequence predicts a peptide of 412 amino acids (aa) including the initiator methionine. There is 98% identity with the aa sequence of the human enzyme. The 3'-untranslated region of the cDNA includes two regions of sequence which are conserved between mouse, rat, human and cow. The murine gene contains 16 exons with the start codon occurring in exon 3. Although alternative splicing occurs in primates to include or exclude exon 2, exon 2 sequences were included in the murine mRNA in all tissues and developmental stages examined. The inclusion of exon 2 in murine mRNA, compared to the usual exclusion in human mRNA, may be explained by differences in the donor splice sequences for exon 2.     

Molecular characterization of equine thymidine kinase 1 and preliminary evaluation of its suitability as a serum biomarker for equine lymphoma

Leucine-rich-repeat receptor-like kinases (LRR-RLKs) play central roles in sensing various signals to regulate plant development and environmental responses. The extracellular domains (ECDs) of plant LRR-RLKs contain LRR motifs, consisting of highly conserved residues and variable residues, and are responsible for ligand perception as a receptor or co-receptor. However, there are few comprehensive studies on the ECDs of LRR-RLKs due to the difficulty in effectively identifying the divergent LRR repeats. In the current study, an efficient LRR motif prediction program, the “Phyto-LRR prediction” program, was developed based on the position-specific scoring matrix algorithm (PSSM) with some optimizations. This program was trained by 16-residue plant-specific LRR-highly conserved segments (HCS) from LRR-RLKs of 17 represented land plant species and a database containing more than 55,000 predicted LRRs based on this program was constructed. Both the prediction tool and database are freely available at http://phytolrr.com/ for website usage and at http://github.com/phytolrr for local usage. The LRR-RLKs were classified into 18 subgroups (SGs) according to the maximum-likelihood phylogenetic analysis of kinase domains (KDs) of the sequences. Based on the database and the SGs, the characteristics of the LRR motifs in the ECDs of the LRR-RLKs were examined, such as the arrangement of the LRRs, the solvent accessibility, the variable residues, and the N-glycosylation sites, revealing a comprehensive profile of the plant LRR-RLK ectodomains. The “Phyto-LRR prediction” program is effective in predicting the LRR segments in plant LRR-RLKs, which, together with the database, will facilitate the exploration of plant LRR-RLKs functions. Based on the database, comprehensive sequential characteristics of the plant LRR-RLK ectodomains were profiled and analyzed.     

Mutants at the Slender1 locus of barley cv Himalaya. Molecular and physiological characterization

A dominant dwarf mutant of barley (Hordeum vulgare) that resembles dominant gibberellin (GA) "-insensitive" or "-nonresponsive" mutants in other species is described. alpha-Amylase production by endosperm half-grains of the mutant required GA3 at concentrations about 100 times that of the WT. The mutant showed only a slight growth response to GA3, even at very high concentrations. However, when additionally dwarfed, growth rate responded to GA3 over the normal concentration range, although only back to the original (dwarf) elongation rate. Genetic studies indicated that the dominant dwarf locus was either closely linked or identical to the Sln1 (Slender1) locus. A barley sequence related to Arabidopsis GAI/RGA was isolated, and shown to represent the Sln1 locus by the analysis of sln1 mutants. The dominant dwarf mutant was also altered in this sequence, indicating that it too is an allele at Sln1. Thus, mutations at Sln1 generate plants of radically different phenotypes; either dwarfs that are largely dominant and GA "-insensitive/-nonresponsive," or the recessive slender types in which GA responses appear to be constitutive. Immunoblotting studies showed that in growing leaves, SLN1 protein localized almost exclusively to the leaf elongation zone. In mutants at the Sln1 locus, there were differences in both the abundance and distribution of SLN1 protein, and large changes in the amounts of bioactive GAs, and of their metabolic precursors and catabolites. These results suggest that there are dynamic interactions between SLN1 protein and GA content in determining leaf elongation rate.     

Molecular characterization of RAPD and SCAR markers linked to the Tm-1 locus in tomato

We have cloned and sequenced six RAPD fragments tightly linked to the Tm-1 gene which confers tomato mosaic virus (ToMV) resistance in tomato. The terminal ten bases in each of these clones exactly matched the sequence of the primer for amplifying the corresponding RAPD marker, except for one in which the 5-endmost two nucleotides were different from those of the primer. These RAPD clones did not cross-hybridize with each other, suggesting that they were derived from different loci. From Southern-hybridization experiments, five out of the six RAPD clones were estimated to be derived from middle- or high-repetitive sequences, but not from any parts of the ribosomal RNA genes (rDNA), which are known to be tightly linked with the Tm-1 locus. The remaining clone appeared to be derived from a DNA family consisting of a few copies. These six RAPD fragments were converted to sequence characterized amplified region (SCAR) markers, each of which was detectable using a pair of primers having the same sequence as that at either end of the corresponding RAPD clone. All pairs of SCAR primers amplified distinct single bands whose sizes were the same as those of the RAPD clones. In four cases, the SCAR markers were present in the line with Tm-1 but absent in the line without it, as were the corresponding RAPD markers. In the two other cases, the products of the same size were amplified in both lines. When these SCAR products were digested with different restriction endonucleases which recognize 4-bp sequences, however, polymorphisms in fragment length were found between the two lines. These co-dominant markers are useful for differentiating heterozygotes from both types of homozygote.     

Molecular characterization and expression of equine testicular cytochrome P450 aromatase

We characterized testicular equine aromatase and its expression. A 2707 bp cDNA was isolated, it encoded a polypeptide of 503 residues with a deduced molecular mass of 57.8 kDa. The sequence features were those of a cytochrome P450 aromatase, with a 78% polypeptide identity with the human counterpart. The gene has a minimal length of 74 kb comprising at least 9 exons and expresses a 2.8 kb mRNA in the testis. Transient cDNA transfections in E293 cells and in vitro translations in a reticulocyte lysate system allowed aromatase protein and activity detections. The activity increased with androstenedione as substrate in a dose-dependent manner. The isolation of testicular aromatase by a new immunoaffinity method demonstrated that the protein could exist either glycosylated or not with a 2 kDa difference. All these results taken together allow new structural studies to progress in the understanding of this cytochrome P450.     

Molecular characterization of a host-range-determining locus from Agrobacterium tumefaciens.

The virulence loci play an essential role in tumor formation by Agrobacterium tumefaciens. This study focused on the virC locus, which affects the host range Agrobacterium species. virC mutants display an attenuated or avirulent phenotype on certain host plants, but remain fully virulent on other plant hosts. The nucleotide sequence revealed that the virC locus of pTiA6NC is an operon consisting of two open reading frames. These two open reading frames, designated virC1 and virC2, encode protein products of 25,713 and 22,710 daltons, respectively, which were visualized by polyacrylamide gel electrophoresis. Only two nucleotides separated the stop codon for virC1 from the start codon for virC2, indicating that these genes may be translationally coupled.     

Molecular characterization of equine herpesvirus 1 (EHV-1) isolated from cattle indicating no specific mutations associated with the interspecies transmission

Interspecies trasmission of equine herpesvirus 1 (EHV-1) from horse to cattle was shown by Crandell et al. (1988). Specific mutations related to the transmission were studied by comparison of five EHV-1 isolates in cattle (BH1247, 3M20-3, G118, G1753, and 9BSV4) using polymerase chain reaction and restriction fragment length polymorphism analysis with added sequencing. G118 and 3M20-3 were the genome type EHV-1 P, while G1753 was the genome type EHV-1 B. BH1247 and 9BSV4 might be other genome types. We could not identify specific mutations related to the interspecies transmission.     

Molecular characterization and expression pattern of the equine lactate dehydrogenase A and B genes

The species-specific properties of LDH isozymes are essentially determined by M (muscle) and H (heart) subunit proteins encoded by the LDHA and LDHB genes, respectively. In the present study, we molecularly characterized the full-length equine lactate dehydrogenase A (eLDHA) and B (eLDHB) cDNAs. The eLDHA cDNA consisted of a 999-bp open reading frame (ORF), while the eLDHB and newly acquired bat LDHB consisted of a 1002-bp ORF, which is 3 bp shorter than the LDHB ORF of other registered mammals. The alignment of amino acid sequences showed that eLDHA acquired positively charged His 88 and 226, and eLDHB lost negatively charged Glu 14, as compared to the highly conserved residues at these positions in the corresponding amino acid sequences of other mammals. These alterations were identified in six equine species by genomic DNA analysis. A comparison of the equine and human 3D structures revealed that the substituted His 88 and 226 of the eLDHA monomer and the deleted Glu 14 of the eLDHB monomer altered the surface charge of equine LDH tetramers and that these three residues were located in important regions affecting the catalytic kinetics. Also, RT-PCR amplification of the three myosin heavy chain isoforms corroborated that the cervical muscle as postural muscle of the thoroughbred horse was composed of more oxidative myofibers than the dynamic muscle. Based on this property, the mRNA expression patterns of eLDHA, eLDHB, and eGAPDH in various tissues were analyzed by using real-time PCR. The expression levels of these three genes in the cervical muscle were not always relatively higher than in the brain or heart.     

Molecular characterization of the S locus in two self-incompatible Brassica napus lines.

In Brassica species, self-incompatibility has been mapped genetically to a single chromosomal location. In this region, there are two closely linked genes coding for the S locus glycoprotein (SLG) and S locus receptor kinase (SRK). They appear to comprise the pistil component of the self-incompatibility reaction. SLG and SRK are thought to recognize an unknown pollen component on the incompatible pollen, and the gene encoding this pollen component must also be linked to the SLG and SRK genes. To further our understanding of self-incompatibility, the chromosomal region carrying the SLG and SRK genes has been studied. The physical region between the SLG-910 and the SRK-910 genes in the Brassica napus W1 line was cloned, and a search for genes expressed in the anther revealed two additional S locus genes located downstream of the SLG-910 gene. Because these two genes are novel and are conserved at other S alleles, we designated them as SLL1 and SLL2 (for S locus-linked genes 1 and 2, respectively). The SLL1 gene is S locus specific, whereas the SLL2 gene is not only present at the S locus but is also present in other parts of the genomes in both self-incompatible and self-compatible Brassica ssp lines. Expression of the SLL1 gene is only detectable in anthers of self-incompatible plants and is developmentally regulated during anther development, whereas the SLL2 gene is expressed in anthers and stigmas in both self-incompatible and self-compatible plants, with the highest levels of expression occurring in the stigmas. Although SLL1 and SLL2 are linked to the S locus region, it is not clear whether these genes function in self-incompatibility or serve some other cellular roles in pollen-pistil functions.     

Molecular characterization of the CAN1 locus in Saccharomyces cerevisiae. A transmembrane protein without N-terminal hydrophobic signal sequence

The complete DNA sequence of the CAN1 locus of the yeast Saccharomyces cerevisiae is presented. The predicted primary translation product consists of 590 amino acids. From the hydropathic profile of the amino acid sequence (as calculated by the algorithm of Kyte and Doolittle (Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132)), one can divide the protein into two distinct regions. The 93-amino acid long N-terminal domain is extremely hydrophilic and does not exhibit any cleavable signal sequence. The rest of the protein (from amino acids 94 to 590) shows features typical for an integral membrane protein. The proposal for the N terminus of the primary translation product is based on results obtained by S1 mapping, insertion mutagenesis, and gene fusion experiments.     

Identification and characterization of the ICP22 protein of equine herpesvirus 1.

The equine herpesvirus 1 (EHV-1) homolog of herpes simplex virus type 1 ICP22 is differently expressed from the fourth open reading frame of the inverted repeat (IR4) as a 1.4-kb early mRNA and a 1.7-kb late mRNA which are 3' coterminal (V. R. Holden, R. R. Yalamanchili, R. N. Harty, and D. J. O'Callaghan, J. Virol. 66:664-673, 1992). To extend the characterization of IR4 at the protein level, the synthesis and intracellular localization of the IR4 protein were investigated. Antiserum raised against either a synthetic peptide corresponding to amino acids 270 to 286 or against a TrpE-IR4 fusion protein (IR4 residues 13 to 150) was used to identify the IR4 protein. Western immunoblot analysis revealed that IR4 is expressed abundantly from an open reading frame composed of 293 codons as a family of proteins that migrate between 42 to 47 kDa. The intracellular localization of IR4 was examined by cell fractionation, indirect immunofluorescence, and laser-scanning confocal microscopy. These studies revealed that IR4 is localized predominantly in the nucleus and is dispersed uniformly throughout the nucleus. Interestingly, when IR4 is expressed transiently in COS-1 or LTK- cells, a punctate staining pattern within the nucleus is observed by indirect immunofluorescence. Cells transfected with an IR4 mutant construct that encodes a C-terminal truncated (19 amino acids) IR4 protein exhibited greatly reduced intranuclear accumulation of the IR4 protein, indicating that this domain possesses an important intranuclear localization signal. Western blot analysis of EHV-1 virion proteins revealed that IR4 proteins are structural components of the virions. Surprisingly, the 42-kDa species, which is the least abundant and the least modified form of the IR4 protein family in infected cell extracts, was the most abundant IR4 protein present in purified virions.     

Isolation and partial characterization of equine herpesvirus type 1 in Czechia

Equine herpesvirus type 1 was determined as the etiological cause of an abortion storm in Czechia in 2003 after the virus strain was isolated from aborted fetus and identified by serological means and by PCR technique. Cloning and sequencing of the glycoprotein D confirmed the identity of the isolates and showed molecular relationships to known EHV-1 strains. Comparison of glycoprotein D sequences with corresponding sequence of EHV-1 reference strains (Kentucky-A and Ab1) revealed high nucleotide homology. The Czech isolate of EHV-1 virus does not differ significantly from the Ab1 strain regarding the glycoprotein D gene and does not bear the frameshift in the 3' terminus which occurs in the Kentucky-A strain.