“Happy mapping” is an in vitro genome mapping method for type 1 and 2 markers that offers several advantages over the RH method. However, it is highly dependent on a whole-genome amplification step that till now has been difficult to realize given the very small amount of starting DNA. In this article, advantages and possible future developments are discussed in light of the difficulties inherent in the method. Keywords: Happy mapping; RH mapping; whole-genome amplification; degenerated PCR; microarrays

The canine species includes over 350 breeds, all different in terms of phenotype, behaviour and disease susceptibility. However, each breed has an extremely homogeneous phenotype and can be considered as a genetic isolate similar to those used by geneticists to identify genes responsible for complex human diseases. Hereditary diseases in dogs very often have an equivalent in man, and certain rare human diseases, sometimes genetically heterogeneous, are found with a high incidence and high specificity in a given dog breed. Given the real difficulty to recruit informative families to determine the genetic causes of complex diseases, dogs offer an alternative to collect data, from animals seen in consultation, usable to identify morbid or susceptibility genes or alleles. This review shows, with several examples, how the determination of genetic causes of hereditary diseases in dogs has powerful and useful applications in human and veterinary medicine.

The Saccharomyces cerevisiae gene PKCl encodes a protein kinase C isozyme that regulates cell wall synthesis. Here we describe the characterization of HOCl, a gene identified by its ability to suppress the cell lysis phenotype of pkcZ-?71 cells. The HOC1 gene (Homologous to OCHl) is predicted to encode a type I1 integral membrane protein that strongly resembles Ochlp, an a-1,6-mannosyltransferase. Immunofluorescence studies localized Hoclp to the Golgi apparatus. While overexpression of HOCl rescued the pkcl-371 temperature-sensitive cell lysis phenotype, disruption of HOCl lowered the restrictive temperature of the pkcl-371 allele. Disruption of HOCl also resulted in hypersensitivity to Calcofluor White and hygromycin B, phenotypes characteristic of defects in cell wall integrity and protein glycosylation, respectively. The function of HOCl appears to be distinct from that of OCHl. Taken together, these results suggest that HOCl encodes a Golgi-localized putative mannosyltransferase required for the proper construction of the cell wall.

We describe here the construction of six deletion mutants and their basic phenotypic analysis. Six open reading frames (ORFs) from chromosome X, YJR039w, YJR041c, YJR043c, YJR046w, YJR053w and YJR065c, were disrupted by deletion cassettes with long (LFH) or short (SFH) flanking regions homologous to the target locus. The LFH deletion cassette was made by introducing into the kanMX4 marker module two polymerase chain reaction (PCR) fragments several hundred base pairs (bp) in size homologous to the promoter and terminator regions of a given ORF. The SFH gene disruption construct was obtained by PCR amplification of the kanMX4 marker with primers providing homology to the target gene. The region of homology to mediate homologous recombination was about 70 bp. Sporulation and tetrad analysis revealed that ORFs YJR041c, YJR046w and YJR065c are essential genes. Complementation tests by corresponding cognate gene clones confirmed this observation. The non-growing haploid segregants were observed under the microscope. The yjr041cΔ haploid cells gave rise to microcolonies comprising about 20 to 50 cells. Most yjr046wΔ cells were blocked after one or two cell cycles with heterogeneous bud sizes. The yjr065cΔ cells displayed an unbudded spore or were arrested before completion of the first cell division cycle with a bud of variable size. The deduced protein of ORF YJR065c, that we named Act4, belongs to the Arp3 family of actin-related proteins. Three other ORFs, YJR039w, YJR043c and YJR053w are non-essential genes. The yjr043cΔ cells hardly grew at 15°C, indicating that this gene is required for growth at low temperature. Complementation tests confirmed that the disruption of YJR043c is responsible for this growth defect. In addition, the mating efficiency of yjr043cΔ and yjr053wΔ cells appear to be moderately a ffected. © 1997 John Wiley & Sons, Ltd.

Actin molecules are major cytoskeleton components of all eukaryotic cells. All conventional actins that have been identified so far are 374-376 amino acids in size and exhibit at least 70% amino acid sequence identity when compared with one another. In the yeast Saccharomyces cerevisiae, one conventional actin gene ACT1 and three so-called actin-related genes, ACT2, ACT3 and ACT5, have been identified. We report here the discovery of a new actin-related gene in this organism, which we have named ACT4. The deduced protein, Act4, of 449 amino acids, exhibits only 33.4%, 26.7%, 23.4% and 29.2% identity to Act1, Act2, Act3 and Act5, respectively. In contrast, it is 68.4% identical to the product of the Schizosaccharomyces pombe Act2 gene and has a similar level of identity to other Sch. pombe Act2 homologues. This places Act4 in the Arp3 family of actin-related proteins. ACT4 gene disruption and tetrad analysis demonstrate that this gene is essential for the vegetative growth of yeast cells. The act4 mutants exhibit heterogenous morphological phenotypes. We hypothesize that Act4 may have multiple roles in the cell cycle.

We have sequenced a 61,989 bp stretch located between genes RAD7 and FIP1 of Saccharomyces cerevisiae chromosome X. This stretch contains 36 open reading frames (ORFs) of at least 100 codons. Fourteen of these correspond to sequences previously published as HIT1, CDC8, YAP17, CBF1, NAT1, RPA12, CCT5, TOR1, RFC2, PEM2, CDC11, MIR1, STE18 and GRR1. The proteins deduced from four ORFs (YJR059w, YJR065c, YJR075w, YJR078w) have significant similarity to proteins of known function from yeast or other organisms, including S. cerevisiae serine/threonine-specific protein kinase, Schizosaccharomyces pombe Act2 protein, S. cerevisiae mannosyltransferase OCH1 protein and mouse indoleamine 2,3-dioxygenase, respectively. Four of the remaining 18 ORFs have similarity to proteins with unknown function, six are weakly similar to other known sequences, while another eight exhibit no similarity to any known sequence. In addition, three tRNA genes have been recognized. Three genes clustered within 22 kb (YJR059w, YJR061w and TOR1) have counterparts arranged within 15 kb on the left arm of chromosome XI. The sequence has been deposited in the Genome Sequence Data Base under Accession Number L47993.

A consortium of European laboratories has been organized to systematically sequence the genome of Saccharomyces cerevisiae. As part of the BIOTECH program aimed at sequencing chromosomes XI and II, we have constructed a total genomic library of yeast strain FY1679 (a direct S288C derivative) into cosmid vectors pWE15 and pOU61cos. Primary clones from four independent libraries totalling 190 genome equivalents have been stored at -80 degrees C. A subset of 1939 independent clones (six genome equivalents) was hybridized using purified chromosomes XI and X as probes. A total of 147 chromosome XI-specific cosmid clones was used to construct the physical map of that chromosome. Mapping methods included a combination of classical bottom-up strategies (fingerprinting, hybridizations) and a novel top-down strategy using I-SceI chromosome fragmentation. The 147 cosmid clones form a unique contig covering the entire chromosome XI (666 kb) with the sole exceptions of the (C1-3A)n repeats of the telomeres. Colinearity of cosmid inserts with yeast DNA was directly verified. A complete EcoRI map of chromosome XI was deduced from partial overlaps of cosmids and used for the sequencing program. Comparison of this map with the genetic map shows unexpected divergences that have been solved by subsequent genetic analysis, yet underline the necessity of independent physical mapping in genome projects.

Hhdman‐T‐cell‐leukemia virus type I (HTLV‐I) is the causative agent of adult T‐cell leukemia/lymphomhd (ATL) and tropical spastic paraparesis/HTLV‐I‐associated myelopathy (TSP/HAM). The different disease outcome may be attributable to subtle mutations leading to modification of viral tropism or infectivity. Initial attempts found a very high level of sequence conservation among all HTLV‐I strains. However, only one complete proviral DNA sequence is reported from a TSP/HAM patient, with a provirus derived from immortalized lymphocytes, which might be expected to be a leukemogenic variant rather than a neutrotropic one. We cloned and sequenced a complete HTLV‐I provirus (HTLV‐I boi ) derived from the uncultured lymphocytes of a sub‐acute post‐transfusional TSP/HAM patient with clonal integration of HTLV‐I. HTLV‐I boi proviral genome is 9033 bp long, and its overall genetic organization is similar to that of the prototype HTLV‐I(ATK), without major deletions or insertions. No premature termination codon was found in the 4 open reading frames of the pX region. Divergence at the nucleotide level of HTLV‐l Boi from the reported full‐length HTLV‐I varies from 1 to 9.4%, and indicates that it corresponds to a cosmopolitan genotype. This study did not identify specific sequences associated with neurotropic strains.

We have sequenced a 42,500 bp stretch located on chromosome X of Saccharomyces cerevisiae between the genes MET3 and CDC8 . This stretch contains 24 open reading frames (ORFs) of at least 100 amino acids. Ten of these correspond to previously published sequences, whereas of the 14 remaining ORFs, only one, GTD892, has significant similarity to proteins from yeast or other organisms. It may belong to the family of ubiquitin–protein ligases and be involved in the ubiquitin‐dependent proteolytic pathway. In addition, three tRNA genes were recognized, two of which had not been hitherto localized. The sequence has been deposited in the Genome Sequence Data Base under Accession Number L36344.

We report here the construction of a complete physical map of the chromosome X of yeast Saccharomyces cerevisiae. Fragments resulting from partial Sau3AI digestion of DNA from a diploid strain derived from S288C were ligated to linearized pWE15, a cosmid vector with T3 and T7 promoters. Another library, made in the cosmid vector pOU61 cos, that lacks T3 and T7 promoters, was also used as a source of target clones. Chromosome-X-specific clones were sorted out by hybridization with radiolabelled pulse-field-gel-purified chromosome X as a probe. Then, 254 cosmids were ordered by walking from one to another by hybridization with end-specific T3 or T7 RNA transcripts as probes. The construction was put to the test by hybridization with a battery of chromosome X gene markers, that showed that the physical map and the genetic map were colinear. The validity of the contig was further strengthened by the results of chromosome nested fractionation with meganuclease I-SceI. An EcoRI restriction map of the contig enabled further verification and measurement of the total length of the contig, that was found to be approximately 700 kb in size. In addition to providing a base for the ongoing yeast genome sequencing project, the physical map can be used to map any sequence belonging to chromosome X.Key Words: EcoRI restrictionphysical mappingT3-T7 walkingyeast Saccharomyces cerevisiae chromosome X

We report here the construction of a complete physical map of the chromosome X of yeast Saccharomyces cerevisiae. Fragments resulting from partial Sau3AI digestion of DNA from a diploid strain derived from S288C were ligated to linearized pWE15, a cosmid vector with T3 and T7 promoters. Another library, made in the cosmid vector pOU61 cos, that lacks T3 and T7 promoters, was also used as a source of target clones. Chromosome-X-specific clones were sorted out by hybridization with radiolabelled pulse-field-gel-purified chromosome X as a probe. Then, 254 cosmids were ordered by walking from one to another by hybridization with end-specific T3 or T7 RNA transcripts as probes. The construction was put to the test by hybridization with a battery of chromosome X gene markers, that showed that the physical map and the genetic map were colinear. The validity of the contig was further strengthened by the results of chromosome nested fractionation with meganuclease I-SceI. An EcoRI restriction map of the contig enabled further verification and measurement of the total length of the contig, that was found to be approximately 700 kb in size. In addition to providing a base for the ongoing yeast genome sequencing project, the physical map can be used to map any sequence belonging to chromosome X.

A DNA fragment covering marmoset interferon gamma (MaIFN-gamma) was cloned from the DNA of peripheral blood leucocytes, sequenced and compared to its human IFN-gamma counterpart. The two nucleotide sequences were found to be highly homologous (90.3%). The position of the exons are directly comparable with those of the human IFN-gamma gene and follows the pattern of structural conservation found elsewhere. The present work makes available recombinant MaIFN-gamma for animal experiments.

Polyclonal antibodies directed against the preS2 and S domains of the woodchuck hepatitis virus (WHV) envelope proteins were prepared using synthetic peptides and fusion polypeptides as immunogens. They were tested by immunoblotting and immunoprecipitation of infected woodchuck sera and lysates of a eukaryotic cell line expressing WHV envelope proteins. Only one anti-peptide serum directed against the preS2 domain was reactive with WHV envelope proteins, recognizing the preS2 and preS1 proteins by their preS2 epitopes. With recombinant fusion proteins we generated several anti-S sera, which recognized all envelope proteins, and anti-preS2 antisera, which recognized the preS proteins. Results obtained with our antisera showed that sera of infected woodchucks lack the low glycosylated form (GP33) of the preS2 protein, unlike human hepatitis B virus.

of the aI/80Kd domain of HE patients with the Spa’/S0a, Spar/sob, and SPCUI/~’ variants, Marchesi et a1 have shown that abnormal tryptic cleavages usually occur in the vicinity of amino acid changes2’ Knowledge of the DNA sequence and exon/intron organization of cloned genomic DNA encoding the CUI domain of Sp has allowed demonstration of the specific nucleotide changes of these m~tations~~.~~ and characterization of other mutations causing HE or HPP.13s’4 Until now the nature of the mutation(s) responsible for the phenotype has remained elusive. However, recent studies have suggested that the variant in HE could result from a defect either in the Sp@ or in the Spa hai in.^'.^^ In this study, we report a white French family in which 12 subjects presented with HPP or HE. The eight subjects that were available for further studies were shown to be heterozygous for an variant. Reconstitution studies using isolated

The in vivo activity of ribozymes designed against mRNA coding for E. coli β-galactosidase was tested both in intramolecular and in intermolecular conditions. When recombinant M13 phage DNA carrying on the same molecule the information for both the ribozyme and the target was transfected into bacterial cells, ribozyme activity was observed. Conversely, a ribozyme coded by a recombinant M13 vector, but targeted against an mRNA transcribed from the F episome including the remaining part of the β-galactosidase gene, was inefficient.