<Emphasis Type="Italic">RT1.L</Emphasis>: a family of MHC class Ib genes of the rat major histocompatibility complex with a distinct promoter structure

RT1.L class I antigens have originally been identified in LEW rats by LEW.1LV3-anti-LEW.1LM1 antisera and have been classified as nonclassical. We report now that LEW.1LV3-anti-LEW.1LM1 antisera react with three different antigens, termed RT1.L1, RT1.L2, and RT1.L3. This was found by serological analysis of a panel of transfectants expressing different class I genes of strain LEW with a LEW.1LV3-anti-LEW.1LM1 antiserum and two monoclonal antibodies (mAbs HT20 and HT21) generated in the same strain combination. The antiserum reacted with all three antigens: the two mAbs with RT1.L1 and RT1.L2, respectively. Sequence analysis showed that the genes encoding RT1.L1, RT1.L2, and RT1.L3 cluster together in a phylogenetic analysis of rat and mouse ₁-₂ sequences and that they share an unusual MHC class I promoter in which Enhancer A and B, as well as the interferon response element (IRE), are missing. Exchange of the promoter in RT1.L2 against the classical RT1.A promoter resulted in high surface expression in appropriate transfectants, indicating that the deviant promoter is responsible for the weak surface expression of the RT1.L2 gene. The very similar promoter structures of RT1.L1 and RT1.L3 are likely to contribute also to the weak expression of these genes. As RT1.L3 maps closely to the deletion in the mutant haplotype lm1, the RT1.L family can be located in the class I region extending from Bat1 to Pou5f1. Different from other allogeneic mAbs detecting known class I molecules encoded by genes of the RT1.C/E region, HT20 and HT21 react with a wide panel of strains carrying different RT1 haplotypes. This suggests that nonclassical class I genes of the RT1.L family are present in most RT1 haplotypes.Nucleotide sequences reported in this paper have been submitted to GenBank with accession numbers AF457139 (RT1.L1), AY397759 (RT1.L2) and AY445668 (RT1.L3)     

Murine MHC class Ib gene,<Emphasis Type="Italic"> H2-M2,</Emphasis> encodes a conserved surface-expressed glycoprotein

We have determined the genomic sequence of H2-M2 in seven haplotypes from nine inbred strains of mice and in five wild-derived haplotypes. Except for the spretus haplotype sp1 with a premature stop codon, we found only limited polymorphism. Four of the five amino acid substitutions in the -helices are at positions that would point out from the antigen-binding groove, indicating that the polymorphism might influence receptor recognition rather than antigen binding. The rat homologue, RT1.M2^lv1, has 89% identity to H2-M2 at the nucleotide level and 91% at the amino acid level, and it also encodes an intact MHC class I glycoprotein. Chimeric proteins with ₁₂ or ₃-transmembrane domains encoded by H2-Q9 were detectable on the surface of transfectants with monoclonal antibodies against Qa2, and the full-length M2 protein, labeled by fusion with green fluorescent protein, was detectable with S19.8 monoclonal antibodies. The H2-M2 protein was thus expressed on the cell surface, even in TAP-deficient RMA-S cells at 37 °C, suggesting that it is TAP-independent. We conclude that H2-M2 is a conserved mouse class Ib gene that is translated to a surface-expressed MHC class I molecule with a function still to be elucidated.The nucleotide sequences reported in this paper have been submitted to GenBank with the accession numbers AY302188–AY302217 for all H2-M2 sequences and AY302218 for RT1.M2, AY326271 for RT1.M2-2, and AY327254 for the RT1.M2 microsatellite marker     

Characterization of the horse (<Emphasis Type="Italic">Equus caballus</Emphasis>)<Emphasis Type="Italic"> IGHA</Emphasis> gene

Nucleotide sequences of the immunoglobulin constant heavy chain genes of the horse have been described for IGHM, IGHG and IGHE genes, but not for IGHA. Here, we provide the nucleotide sequence of the genomic IGHA gene of the horse (Equus caballus), including its secretion region and the transmembrane exon. The equine IGHA gene shows the typical structure of a mammalian IGHA gene, with only three exons, separated by two introns of similar size. The hinge exon is located at the 5 end of the CH2 exon and encodes a hinge region of 11 amino acids, which contains five proline residues. The coding nucleotide sequence of the secreted form of the equine IGHA gene shares around 72% identity with the human IGHA1 and IGHA2 genes, as well as the bovine, ovine, porcine and canine IGHA genes, without distinct preference for any of these species. The same species also cluster together in a phylogenetic tree of the IGHA coding regions of various mammals, whereas rodent, rabbit, marsupial and monotreme IGHA genes each build a separate cluster.The nucleotide sequences reported in this paper have been assigned the EMBL/GenBank accession numbers AY247966 and AY351982     

Role of nonclassical class I genes of the chicken major histocompatibility complex Rfp-Y locus in transplantation immunity

The chicken major histocompatibility complex (MHC) genes are organized into two genetically independent clusters which both possess class I and class II genes: the classical B complex and the Restriction fragment pattern-Y (Rfp-Y) complex. In this study, we have examined the role of Rfp-Y genes in transplantation immunity. For this we used three sublines, B19H1, B19H2 and B19H3, derived from a line fixed for B19. Southern blots, PCR-SSCP assays using primers specific for Rfp-Y genes, and Rfp-Y class I allele-specific sequencing show that the polymorphisms observed in B19H1, B19H2 and B19H3 are due to the presence of three different Rfp-Y haplotypes. The Rfp-Y class I (YF) alleles in these three haplotypes are highly polymorphic, and RT-PCR shows that at least two YF loci are expressed in each subline. The three sublines show Rfp-Y-directed alloreactivity in that Rfp-Y-incompatible skin grafts are rejected within 15 days, a rate intermediate between that seen in B-incompatible rejection (7 days) and that observed for grafts within the sublines (20 days). We conclude that Rfp-Y has an intermediate role in allograft rejection, likely to be attributable to polymorphism at the class I loci within this region.The sequence data reported are available in the GenBank database under the accession numbers AY257165 (YFVw*15), AY257166 (YFVw*16), AY257167 (YFVIw*15), AY257168 (YFVIw*17), AY257169 (YFw*16), and AY257170 (YFw*17)     

A rapid identification method for aflatoxin-producing strains ofAspergillus flavus andA. parasiticus by ammonia vapor

The colony reverse of aflatoxin (AF)-producing strains ofAspergillus flavus andA. parasiticus turned pink when their cultures were exposed to ammonia vapor. The color change was visible for colonies grown on media suitable for AF production such as potato dextrose, coconut, and yeast extract sucrose agars after 2 d incubation at 25°C. Of the 120 strains ofA. flavus, A. parasiticus, and two related species inA. flavus group:A. oryzae andA. sojae tested in this study, only the AF-producing strains ofA. flavus andA. parasiticus showed the pink pigmentation. The color change occurred immediately after the colony was contacted with ammonia vapor. This method was useful for rapid screening the AF-producing strains ofA. flavus andA. parasiticus.     

Es-6, a further polymorphic esterase in the rat

A further polymorphic rat esterase with broad tissue expression and restricted substrate specificity is described and tentatively called Es-6. Inbred rat strains have either fixed allele Es-6^F or fixed allele Es-6^S. Es-6 is not linked to the established esterase cluster consisting of the eight esterase loci Es-1, Es-2, Es-3M, Es-4M, Es-4W, Es-5 (=Es-3W), Es-7, and Es-8 in LG V of the rat or to RT1, Gc, c, a, and h. Esterases with apparently identical biochemical and genetical characteristics are Es-17 of the mouse and Es-A4 of humans.Supported by the Deutsche Forschungsgemeinschaft (Be 352/13 and Gu 105).     

Physical map and expression profile of genes of the telomeric class I gene region of the rat MHC

The rat is an important model for studying organ graft rejection and susceptibility to certain complex diseases. The MHC, the RT1 complex, plays a decisive role in controlling these traits. We have cloned the telomeric class I region of the RT1 complex, RT1-C/E/M, of the BN inbred rat strain in a contig of overlapping P1-derived artificial chromosome clones encompassing approximately 2 Mb, and present a physical map of this MHC region. Forty-five class I exon 4-hybridizing BAM:HI fragments were detected, including the previously known rat class I genes RT1-E, RT-BM1, RT1-N, RT1-M2, RT1-M3, and RT1-M4. Twenty-six non-class I genes known to map to the corresponding part of the human and mouse MHC were tested and could be fine mapped in the RT1-C/E/M region at orthologous position. Four previously known microsatellite markers were fine mapped in the RT1-C/E/M region and found to occur in multiple copies. In addition, a new, single-copy polymorphic microsatellite has been defined. The expression profiles of several class I genes and the 26 non-class I genes were determined in 13 different tissues and exhibited restricted patterns in most cases. The data provide further molecular information on the MHC for analyzing disease susceptibility and underline the usefulness of the rat model.     

Actinophages and restriction enzymes fromMicromonospora species (Actinomycetales)

Summary To develop a screening procedure for the detection of restriction endonucleases in micromonosporae and catellatosporae based on efficiency of plating, eight different actinophages were isolated from soils enriched withMicromonospora species and one fromCatellatospora-enriched soil. The lytic actinophages all contained double-stranded DNA and the majority appeared, when examined by electron microscopy, to belong to Ackermann's type B1 since they had isometric heads and noncontractile tails. One actinophage was classified as type C1 because of its isometric head and very short noncontractile tail. The host ranges of the actinophages were determined on strains ofMicromonospora and selected species from other actinomycete genera of cell wall chemotype II. Type II restriction enzymes were isolated fromM. echinospora ssp.echinospora (ATCC 15837),M. purpurea (ATCC 15835) andM. zionensis (LL-100-125) and were designatedMecI,MpuI andMziI, respectively. Restriction enzymesMecI andMpuI are isoschizomers ofXhoI, whileMziI is an isoschizomer ofPvuII.     

Sequencing and analysis of aMycoplasma gallisepticum A5969 chromosome region containing the S10 andrrn23-5 operons

A 20,115-nt region of theMycoplasma gallisepticum A5959 genome was sequenced (GenBank accession no. AF036708). The region contains therrn23-5 and S10 operons, the lactate dehydrogenase gene, and two open reading frames (ORF293 and ORF129/ORF171) coding for proteins of unknown function. Therrn23-5 operon includes genes for 23S and 5S rRNAs. The S10 operon includes genes for 20 ribosomal proteins, Sec Y transport protein, adenylate kinase, and methionine aminopeptidase, and lacks theinfA-rpl36-rps13-rpoA-rpl17 genes found in the S10 operon ofM. genitalium, M. pneumoniae, andBacillus subtilis. The product ofM. gallisepticum ldh is equally similar to the corresponding proteins of mycoplasmata andB. subtilis but contains only a part of the motif characteristic of the active center of lactate dehydrogenases. The chromosome region adjacent to the sequenced one containsuvrA,nrdE,nrdF, andptsI.     

Differentiation ofMelampsora rust species on willows in Japan using PCR-RFLP analysis of ITS regions of ribosomal DNA

To develop a reliable method for identifyingMelampsora species parasitic on willows in Japan, we differentiated 10Melampsora species by PCR-RFLP analysis. Internal transcribed, spacer (ITS) regions, including 5.8S ribosomal DNA, of 63 collections of 10Melampsora species and 4 collections of unidentified species were amplified by PCR. The fragments from the 67 collections varied in size (approximately 880 bp, 860 bp and 840 bp). The restriction sites in the amplified DNA fragments were mapped after the RFLP analysis using four restriction enzymes,Dra I,EcoRI,SspI andTaqI. All the collections were divided into 11 RFLP types. In the 6 species,M. caprearum, M. epiphylla, M. kamikotica, M. larici-urbaniana, M. microsora andM. yezoensis, the RFLP type was species-specific. The RFLP type ofM. chelidonii-pierotii andM. coleosporioides was identical. The collections ofM. epitea were separated into three RFLP types. One of these three types was identical with the type ofM. humilis. It is suggested that the PCR-RFLP analysis of ITS regions is a useful and reliable method for species identification ofMelampsora. Contribution No. 131, Laboratories of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba.     

<Emphasis Type="Italic">AOM3</Emphasis> and<Emphasis Type="Italic"> AOM4</Emphasis>: two MADS box genes expressed in reproductive structures of<Emphasis Type="Italic"> Asparagus officinalis</Emphasis>

The MADS box genes participate in different steps of vegetative and reproductive plant development, including the most important phases of the reproductive process. Here we describe the isolation and characterisation of two Asparagus officinalis MADS box genes, AOM3 and AOM4. The deduced AOM3 protein shows the highest degree of similarity with ZAG3 and ZAG5 of maize, OsMADS6 of rice and AGL6 of Arabidopsis thaliana. The deduced AOM4 protein shows the highest degree of similarity with AOM1 of asparagus, the SEP proteins of Arabidopsis and the rice proteins OsMADS8, OsMADS45 and OsMADS7. The high level of identity between AOM1 and AOM4 made impossible the preparation of probes specific for one single gene, so the hybridisation signal previously described for AOM1 is probably due to the expression of both genes. The expression profile of AOM3 and AOM1/AOM4 during flower development is identical, and similar to that of the SEP genes. Asparagus genes, however, are expressed not only in flower organs, but also in the different meristem present on the apical region of the shoot during the flowering season: the apical meristem and the three lateral meristems emerging from the leaf axillary region that will give rise to flowers and lateral inflorescences during flowering season, and to phylloclades and branches during the subsequent vegetative phase. The expression of AOM3 and AOM1/AOM4 in these meristems appears to be correlated with the reproductive function of the apex as the hybridisation signal disappears when the apex switches to vegetative function.     

Selective antimicrobial action of chitosan against spoilage yeasts in mixed culture fermentations

The effect of chitosan on Saccharomyces cerevisiae (the yeast that carries out alcohol fermentation), Brettanomyces bruxellensis and Brettanomyces intermedius (contaminants of alcohol fermentations), was investigated. The effect of chitosan was tested on each yeast, as well as on mixed cultivations of S. cerevisiae + B. bruxellensis and S. cerevisiae + B. intermedius. Chitosan enhanced the lag period of both strains of Brettanomyces (80 h for B. bruxellensis and 170 h for B. intermedius with 6 and 2 g/l chitosan, respectively). The growth rate of S. cerevisiae was inversely proportional to the chitosan concentration; the former was 50% when 6 g/l polysaccharide was used. Moreover, in mixed cultivations of S. cerevisiae and Brettanomyces strains, it was found that both B. bruxellensis and B. intermedius failed to grow while growth of S. cerevisiae was not affected (using 3 and 6 g/l chitosan, respectively). An interesting collateral result was that the presence of chitosan accelerated the consumption of glucose in the mixed cultivations (60 h instead of 120 h).     

The karyotypes ofMicroseris bigelovii,M. douglasii,andM. pygmaea (Asteraceae)

The karyotypes of the three annuals,Microseris bigelovii, M. douglasii andM. pygmaea, consist of 2n = 18, small, submetacentric chromosomes. Length, centromere position, C-banding pattern, silver staining of NOR's, and the use of base specific fluorochromes, allow the identification of four of the nine chromosome pairs. The banding pattern ofM. bigelovii andM. pygmaea is identical, but intraspecific differences are found between strains ofM. douglasii.     

Light induction of the clock-controlled geneccg-1 is not transduced through the circadian clock inNeurospora crassa

Ambient light and the circadian clock have been shown to be capable of acting either independently or in an interrelated fashion to regulate the expression of conidiation in the ascomycete fungusNeurospora crassa. Recently several molecular correlates of the circadian clock have been identified in the form of the morning-specific clock-controlled genesccg-1 andccg-2. In this paper we report studies on the regulation ofccg-1, an abundantly expressed gene displaying complex regulation. Consistent with an emerging consensus for clock-controlled genes and conidiation genes inNeurospora, we report thatccg-1 expression is induced by light, and show that this induction is independent of the direct effects of light on the circadian clock. Although circadian regulation of the gene is lost in strains lacking a functional clock, expression ofccg-1 is still not constitutive, but rather fluctuates in concert with changes in developmental potential seen in such strains. Light induction ofccg-1 requires the products of theNeurospora wc-1 andwc-2 genes, but surprisingly the requirement forwc-2 is suppressed in conditional mutants ofcot-1, a gene that encodes a cAMP-dependent protein kinase. These data provide insight into a complex regulatory web, involving at least circadian clock control, light control, metabolic control, and very probably developmental regulation, that governs the expression ofccg-1.     

DNA evidence for multiple origins of intergeneric allopolyploids in annualMicroseris (Asteraceae)

Seventy populations of North American annualMicroseris, Stebbinsoseris, andUropappus species were examined for chloroplast and nuclear ribosomal DNA restriction site variability to determine the origin of the allotetraploid speciesS. heterocarpa andS. decipiens. Previously identified chloroplast DNA restriction site variants were used in concert with restriction site variation forNco I in the nuclear-encoded ribosomal DNA repeat. The presence of two, mutually exclusive restriction site gains were observed in diploid populations ofM. douglasii; these same variants were also found in populations of allotetraploidS. heterocarpa, indicating mutiple origins of this species from different maternal diploid populations ofM. douglasii. Variation in the rDNA repeat between the diploid annual species and the putative paternal genome ofU. lindleyi was found to be additive inS. heterocarpa. A similar relationship was observed for the origin ofS. decipiens; cpDNA restriction site variants found inM. bigelovii andM. douglasii were present inS. decipiens. The rDNANco I variants also were additive in this purported allotetraploid. These results confirm the reticulate evolutionary pattern inStebbinsoseris and provide another example of multiple origins of intergeneric allopolyploids.     

The immunoglobulin <Emphasis Type="Italic">IGHD</Emphasis> gene locus in C57BL/6 mice

Four immunoglobulin heavy chain diversity (IGHD) gene subgroups (DFL16, DSP2, DQ52, and DST4) have been identified previously in BALB/c mice. Although the locations of most IGHD genes have been established based on restriction map and Southern blot analysis, a complete mouse IGHD gene locus map at the sequence level is still not available. In addition, a previous restriction fragment length polymorphism study suggested that significant difference in the IGHD gene locus exists between C57BL/6 and BALB/c mice. The author has now analyzed the C57BL/6 mouse genomic data and established a complete map of the IGHD gene locus. All four IGHD subgroups previously identified in BALB/c mice were found to be present in C57BL/6 mice. However, unlike the BALB/c mice, which have at least 13 IGHD genes, the C57BL/6 genome contains only ten IGHD genes, which include one DFL16, six DSP2, one DQ52, and two DST4 genes. There are also differences in the coding regions of the DST4 and DQ52 genes between the two mouse strains.