A divergent non-classical class I gene conserved in salmonids

 Complementary DNA for two class I genes of the rainbow trout, Oncorhynchus mykiss, were characterized. MhcOnmy-UBA*01 is similar to Onmy-UA-C32 and the classical major histocompatibility complex class I genes of other fish species, whereas Onmy-UAA*01 is divergent from all class I genes so far characterized. Onmy-UAA*01 is expressed at lower levels than Onmy-UBA*01. Although Onmy-UAA*01 exhibits restriction fragment length polymorphism on Southern blotting, the encoded protein is highly conserved. Two allotypes, which differ only by substitution at amino acid position 223 of the α₃ domain, have been defined. Onmy-UAA*01 has an exon-intron organization like other class I genes and contains a Tc1-like transposon element in intron III. Orthologues of Onmy-UAA*01 have been characterized in four other species of salmonid. Between four species of Oncorhynchus, UAA*01 proteins differ by only 2–6 amino acids, whereas comparison of Oncorhynchus with Salmo trutta (brown trout) reveals 14–16 amino acid differences. The Onmy-UAA*01 gene has properties indicative of a particularly divergent non-classical class I gene. Received: 22 September 1998 / Revised: 24 November 1998     

Repression of the expression of genes encoding xylanolytic enzymes in Aspergillusoryzae by introduction of multiple copies of the xynF1 promoter

A xylanase gene, xynF1, was cloned and characterized from a shoyu koji mould Aspergillus oryzae KBN616. The xynF1 gene was found to be comprised of 1484 bp with ten introns. The deduced amino acid sequence encodes a protein consisting of 327 amino acids (35,402 Da) which is very similar to the fungal family F xylanases such as Aspergillus nidulans XlnC, Aspergillus kawachii XynA and Penicillium chrysogenum XylP. The intron/exon organization of xynF1 is very similar to that of the fungal family F xylanase genes. Plasmid pXPR64, which contains 64 copies of the xynF1 promoter region (PxynF1) in the same direction, was constructed and introduced into A. oryzae. This led to reduced expression of both xylanase and β-xylosidase genes in the transformants. Received: 18 May 1998 / Received revision: 7 July 1998 / Accepted: 9 July 1998     

Localization of five new Ly49 genes, including three closely related to Ly49c

 Nine genes belonging to the mouse Ly49 multigene family of natural killer cell receptors have been identified to date. Two of these genes, Ly49h and i, are very closely related to the well characterized Ly49c gene in the carbohydrate recognition domain. Here we show by Southern blotting that at least two additional new sequences exist in C57BL/6 mice that are also closely related to Ly49c in the carbohydrate recognition domain. Furthermore, in contrast to Ly49a, extensive variation in the arrangement and number of Ly49c–related genes in different mouse strains was observed. To characterize and localize the new Ly49c–related genes in C57BL/6 mice, we isolated and mapped genomic P1 clones hybridizing to an Ly49C exon 7 probe. Locations and the relative order of all Ly49 genes found within the clones was determined. We also used polymerase chain reaction to sequence exons 2, 4, and 7 from all genes. In this manner, we identified five new potential Ly49 genes which have been tentatively termed Ly49j-n. Ly49j, k, and n belong to the Ly49c–related subfamily, whereas Ly49l and Ly49m are most similar to Ly49d and g, respectively. Interestingly, the members of the Ly49c–related subfamily are not clustered as a unit but are interspersed among other Ly49 genes. These results illustrate the complex nature of the Ly49 gene family and should aid in the understanding of functions, such as the mediation of hybrid resistance, in which Ly49c–related genes play a role. Received: 10 December 1997 · Revised: 28 February 1998     

Identification of 1-aminocyclopropane-1-carboxylic acid synthase genes controlling the ethylene level of ripening fruit in Japanese pear (Pyrus pyrifolia Nakai)

The shelf life of Japanese pear fruit is determined by its level of ethylene production. Relatively high levels of ethylene reduce storage potential and fruit quality. We have identified RFLP markers tightly linked to the locus that determines the rate of ethylene evolution in ripening fruit of the Japanese pear. The study was carried out using sequences of two types of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase genes (PPACS1 and pPPACS2) and a ACC oxidase gene (PPAOX1) as probes on 35 Japanese pear cultivars expressing different levels of ethylene (0.0∼300 μl/kg fresh weight/h) in ripening fruit. When total DNA was digested with HindIII and probed with pPPACS1, we identified a band of 2.8 kb which was specific to cultivars having very high ethylene levels (≧10 μ1/kg f.w./h) during fruit ripening. The probe pPPACS2 identified a band of 0.8 kb specific to cultivars with moderate ethylene levels (0.5 μl/kg f.w./h–10 μl/kg f.w./h) during fruit ripening. The cultivars that produce high levels of ethylene possess at least one additional copy of pPPACS1 and those producing moderate levels of ethylene have at least one additional copy of pPPACS2. These results suggest that RFLP analysis with different ACC synthase genes could be useful for predicting the maximum ethylene level during fruit ripening in Japanese pear. Received: 1 July 1998 / Accepted: 6 October 1998     

Genetic analysis of mutant clones of Chlamydomonas reinhardtii defective in potassium transport

Mutant clones of Chlamydomonas reinhardtii defective for potassium transport were isolated and characterized. Of the four genes identified, three –TRK1, TRK2 and TRK3– encode high-affinity transport functions, and one gene, HKR1, encodes a low-affinity transport function. Characterization of the potassium dependence of recombinants possessing two mutant trk alleles suggests that the protein products of TRK2 and TRK3 interact functionally, and that TRK1 may serve a regulatory function. The mutant clone defective for a low-affinity potassium transporter was isolated by mutagenizing trk2-1 cells, which lack a functional high-affinity transporter, and screening surviving cells for dependence on very high potassium concentrations. The hkr1 phenotype is expressed only in the presence of trk2-1. Received: 24 August 1998 / Accepted: 16 November 1998     

IL1B gene polymorphisms influence the course and severity of inflammatory bowel disease

 There is evidence of a disbalance in the inflammatory regulation of patients with inflammatory bowel diseases (IBD). Interleukin-1β plays an important role in the pro-inflammatory response. Our aim was to study the influence which IL1B gene polymorphisms may have on the severity and course of these diseases. Ninety-six patients with ulcerative colitis (UC), 98 patients with Crohn's disease (CD), and 132 ethnically matched healthy individuals (HC) were typed for the polymorphic sites in the promoter region (position –511) and in exon 5 (position +3953) of the IL1B gene, using polymerase chain reaction (PCR)-based methods. In the CD group a significant association (P=0.009) was found in this pair of genes. Homozygotes for allele 1 at position +3953 were more often present (69% vs 31%) in the subgroup of patients carrying at least one copy of allele 2 at position –511. This association was significant in patients with non-perforating disease (P=0.002), but was not present in patients with perforating-fistulizing disease. The distribution of both allelic pairs in the non-fistulizing group proved to be significantly different from HC (P<0.05), UC (P<0.03), and the fistulizing group (P<0.05). There was a similar association in non-operated patients (P=0.024), whereas no such association was found in surgically treated patients. Among carriers of allele 2 at position –511, UC patients with more severe bleeding symptoms (P=0.006) were less frequently found. These results suggest that IL1B gene polymorphisms participate in determining the course and severity of inflammatory bowel disease and contribute to explain the heterogeneity of these diseases. Received: 16 July 1998 / Revised: 3 November 1998     

What is a bona fide mating-type gene? Internuclear complementation of mat mutants in Podospora anserina

In the heterothallic ascomycete Podospora anserina, the mating-type locus is occupied by two mutually exclusive sequences termed mat+ and mat–. The mat+ sequence contains only one gene, FPR1, while the mat– sequence contains three genes: FMR1, SMR1 and SMR2. Previous studies have demonstrated that FPR1 and FMR1 are required for fertilization. Further analyses have led to the hypothesis that mat+ and mat– genes establish a mat+ and mat– nuclear identity, allowing recognition between nuclei of opposite mating type within the syncytial cells formed after fertilization. This hypothesis was based on the phenotypes of strains bearing mutations in ectopic mat genes. Here we present an analysis of mutations in resident mat– genes which suggests that, unlike FMR1 and SMR2, SMR1 is not involved in establishing nuclear identity. In fact, mutations in these two genes impair nuclear recognition, leading to uniparental progeny, while mutations in SMR1 block the sexual process, probably at a step after nuclear recognition. The nuclear identity hypothesis has also been tested through internuclear complementation tests. In these experiments, the mat– mutants were crossed with a mat+ strain carrying the wild-type mat– genes. Our rationale was that internuclear complementation should not be possible for nuclear identity genes: the relevant genes should show nucleus-restricted expression, and diffusion of their products to other nuclei should not occur. This test confirmed that SMR1 is not a bona fide mat gene since it can fulfill its function whatever its location, in either a mat− or a mat+ nucleus, and even when present in both nuclei. SMR2, but not FMR1, behaves like a nuclear identity gene with respect to internuclear complementation tests. A model is proposed that tentatively explains the ambiguous behaviour of the FMR1 gene and clarifies the respective functions of the three mat– proteins. Received: 15 October 1996 / Accepted: 25 April 1997     

DNA sequence analysis of the C4 antigen WH: evidence for two mechanisms of expression

 Amino acid and protein analyses have allowed the construction of a model for the C4-based Rodgers and Chido blood group antigens. The single low-frequency allele (WH) in this blood group system, however, has not been characterized at the molecular level. Two WH⁺ donors were studied by C4 agarose gel electrophoreses, immunoblot studies using monoclonal anti-Rg: 1 or anti-Ch: 1, serological phenotyping, polymerase chain reaction-restriction fragment length polymorphism of their C4 genes, and DNA sequencing of the WH allele. The first donor had the C4A1, A3 phenotype; the C4A1 carried Ch: 1, 3, 6 (thus exhibiting reversed antigenicity) and the C4A3 carried the WH antigen. The amino acid sequence of the WH allele was PCPVLD at positions 1101 – 1106, S at position 1157, and VDLL at positions 1188 – 1191. A second donor typed as C4A2, A4, B1 and was also WH⁺. Immunoblot analysis showed that a C4B1 protein expressed Rg: 1. Sequence analysis of the C4B genes showed the amino acids LSPVIH at positions 1101 – 1106, S at position 1157, and ADLR at positions 1188 – 1191. Thus, the WH antigen is a conformational epitope that can arise through different mechanisms on either a C4A or C4B gene. Received: 22 November 1995 / Revised: 19 February 1996     

Molecular characterization of pdc2 and mapping of three pdc genes from rice

 The anaerobic fermentation pathway is thought to play an important role under flooding conditions. The pyruvate decarboxylase 2 (pdc2) gene that encodes the first enzyme of this pathway has been cloned and characterized from rice. This gene has an open reading frame that putatively encodes a 603 amino-acid-residue protein with a molecular mass of 64 kDa. pdc2 has five introns dispersed throughout the coding region, which is also true for rice pdc1. Although the length of these introns in rice pdc2 are different from those in rice pdc1, they are located in exactly the same positions based on the deduced amino-acid sequences. The temporal and spatial expression patterns of pdc1 and pdc2 show that pdc2 is induced to a higher level during the early period (1.5–12 h) of anoxia than pdc1, which is induced more after longer time periods (24–72 h) of anoxia in both shoots and roots. The map positions of the three pdc genes have also been determined. Rice pdc1 is located on chromosome 5 between BCD454A and RZ67, pdc2 is located on chromosome 3 between RZ329 and RZ313, and pdc3 is mapped on chromosome 7 distal to RG351. Received: 19 May 1998 / Accepted: 29 September 1998     

Genomic structure and chromosome location of the murine PDE1B phosphodiesterase gene

Cyclic nucleotide phosphodiesterases (PDEs) catalyze the hydrolysis of cAMP and cGMP, thereby participating in regulation of the intracellular concentrations of these second messengers. The PDE1 family is defined by regulation of activity by calcium and calmodulin. We have cloned and characterized the mouse PDE1B gene, which encodes the 63-kDa calcium/calmodulin-dependent PDE (CaM-PDE), an isozyme that is expressed in the CNS in the olfactory tract, dentate gyrus, and striatum and may participate in learning, memory, and regulation of phosphorylation of DARPP-32 in dopaminergic neurons. We screened an I-129/SvJ mouse genomic library and identified exons 2–13 of the PDE1B gene that span 8.4 kb of genomic DNA. Exons range from 67 to 205 nucleotides and introns from 91 to 2250 nucleotides in length. Exon 1 was not present in the 3 kb of genomic DNA 5′ to exon 2 in our clones. The mouse PDE1B gene shares many similar or identical exon boundaries as well as considerable sequence identity with the rat PDE4B and PDE4D genes and the Drosophila dunce cAMP-specific PDE gene dnc, suggesting that these genes all arose from a common ancestor. Using fluorescence in situ hybridization, we localized the PDE1B gene to the distal tip of mouse Chromosome (Chr) 15. Received: 10 November 1997 / Accepted: 12 March 1998