G. Stranzinger

Physically mapped, cosmid-derived microsatellite markers as anchor loci on bovine chromosomes

To identify physical and genetic anchor loci on bovine chromosomes, 13 cosmids, obtained after the screening of partial bovine cosmid libraries with the (CA)n microsatellite motif, were mapped by fluorescence in situ hybridization (FISH). Eleven cosmid probes yielded a specific signal on one of the bovine chromosomes and identified the following loci: D5S2, D5S3, D6S3, D8S1, D11S5, D13S1, D16S5, D17S2, D19S2, D19S3, D21S8. Two cosmids produced centromeric signals on many chromosomes. The microsatellite-containing regions were subcloned and sequenced. The sequence information revealed that the two centromeric cosmids were derived from bovine satellites 1.723 and 1.709, respectively. A cosmid located in the subtelomeric region of Chromosome (Chr) 17 (D17S2) had features of a chromosome-specific satellite. Primers were designed for eight of the nonsatellite cosmids, and seven of these microsatellites were polymorphic with between three and eight alleles on a set of outbred reference families. The polymorphic and chromosomally mapped loci can now be used to physically anchor other bovine polymorphic markers by linkage analysis. The microsatellite primers were also applied to DNA samples of a previously characterized panel of somatic hybrid cell lines, allowing the assignment of seven microsatellite loci to defined syntenic groups. These assignments confirmed earlier mapping results, revealed a probable case of false synteny, and placed two formerly unassigned syntenic groups on specific chromosomes.

Two α(1,2) fucosyltransferase genes on porcine Chromosome 6q11 are closely linked to the blood group inhibitor (S) and Escherichia coli F18 receptor (ECF18R) loci

Abstract. The Escherichia coli F18 receptor locus (ECF18R) has been genetically mapped to the halothane linkage group on porcine Chromosome (Chr) 6. In an attempt to obtain candidate genes for this locus, we isolated 5 cosmids containing the α(1,2)fucosyltransferase genes FUT1, FUT2, and the pseudogene FUT2P from a porcine genomic library. Mapping by fluorescence in situ hybridization placed all these clones in band q11 of porcine Chr 6 (SSC6q11). Sequence analysis of the cosmids resulted in the characterization of an open reading frame (ORF), 1098 bp in length, that is 82.3% identical to the human FUT1 sequence; a second ORF, 1023 bp in length, 85% identical to the human FUT2 sequence; and a third FUT-like sequence thought to be a pseudogene. The FUT1 and FUT2 loci therefore seem to be the porcine equivalents of the human blood group H and Secretor loci. Direct sequencing of the two ORFs in swine being either susceptible or resistant to adhesion and colonization by F18 fimbriated Escherichia coli (ECF18) revealed two polymorphisms at bp 307 (M307) and bp 857 (M857) of the FUT1 ORF. Analysis of these mutations in 34 Swiss Landrace families with 221 progeny showed close linkage with the locus controlling resistance and susceptibility to E. coli F18 adhesion and colonization in the small intestine (ECF18R), and with the locus of the blood group inhibitor S. A high linkage disequilibrium of M307–ECF18R in Large White pigs makes the M307 mutation a good marker for marker-assisted selection of E. coli F18 adhesion-resistant animals in this breed. Whether the FUT1 or possibly the FUT2 gene products are involved in the synthesis of carbohydrate structures responsible for bacterial adhesion remains to be determined.

A DNA polymorphism influencing α(1,2)fucosyltransferase activity of the pig FUT1 enzyme determines susceptibility of small intestinal epithelium to Escherichia coli F18 adhesion

Abstract. The α(1,2)fucosyltransferases (FUT1 and FUT2) contribute to the formation of blood group antigen structures, which are present on cell membranes and in secretions. In the present study we demonstrate that both FUT1 and FUT2 are expressed in the pig small intestine. FUT1 polymorphisms influence adhesion of F18 fimbriated Escherichia coli (ECF18) to intestinal mucosa, and FUT2 is associated with expression of erythrocyte antigen 0. The FUT1 polymorphisms result in amino acid substitutions at positions 103 (Ala→Thr) and 286 (Arg→Glu). Tightly controlled expression of the FUT2 gene results in either an abundance or an absence of mRNA in small intestinal mucosa. ECF18-resistant animals were shown to be homozygous for threonine at amino acid 103 of the FUT1 enzyme. Susceptibility to ECF18 adhesion appeared to be solely dependent on the activity of FUT1 in intestinal epithelia. In intestinal mucosae of ECF18-resistant pigs which expressed FUT1 but not FUT2 RNA, the levels of α(1,2)fucosyltransferase activity were significantly lower (28- to 45-fold, P<0.001) than in susceptible pigs. Moreover, lysates of CHO cells transfected with FUT1 constructs encoding threonine at amino acid position 103 also showed significantly reduced enzyme activity compared with constructs encoding alanine at this position. Our genetic and enzymatic studies support the hypothesis that the FUT1 enzyme, and particularly the amino acid at position 103, is likely important in the synthesis of a structure that enables adhesion of ECF18 bacteria to small intestinal mucosa.

Tumor regression induced by intratumoral injection of DNA coding for human interleukin 12 into melanoma metastases in gray horses

Preclinical studies investigating new therapeutic principles against melanoma are presently being carried out in mouse models; however, these are not optimal. Here we describe a novel animal model using gray horses. These animals spontaneously develop metastatic melanoma that resembles human disease and is thus highly relevant for preclinical studies testing new immunotherapy protocols. We found that injection of plasmid DNA coding for the human cytokine interleukin 12 into established metastases induced significant regression in all 12 treated lesions in a total of 7 horses. Complete disappearance was observed in one treated lesion, with no recurrence after 6xa0months. No adverse events have been observed in any of the animals during and after treatment. These results demonstrate the effectiveness and safety of interleukin 12 encoding plasmid DNA therapy against established metastatic disease in a large animal model and serve as a basis for a clinical trial.

Mapping of bovine cytokeratin sequences to four different sites on three chromosomes

The chromosomal location of bovine class I and class II cytokeratin sequences was determined using in situ hybridization and Southern blot hybridization to DNA from hybrid somatic cells. The main signals were found over chromosome region 19q16----qter after in situ hybridization with two probes for the class I cytokeratin gene subfamily (KRT10 and KRT19) and over region 5q14----q23 after hybridization with probes for the class II gene subfamily (KRT1, KRT5, and KRT8). These regions most likely contain the loci of functional cytokeratin genes, with KRT10 and KRT19 mapping to 19q21 and KRT1, KRT5, and KRT8 to 5q21. The in situ hybridization data were corroborated by analysis of a somatic hybrid cell panel. The genes for the class I keratins segregated concordantly with each other and syntenic group U21 but were discordant with the class II keratin genes. The class II keratin genes segregated concordantly with each other and syntenic group U3. Two class II gene probes gave an additional minor signal above chromosome region 5q25----q33 after in situ hybridization, while another class II probe yielded a minor signal above chromosome region 10q31----qter. When the latter probe and an additional linked probe were hybridized to DNAs from a hybrid panel, two independently segregating loci were recognized, one of which cosegregated with the class II subfamily in syntenic group U3 and the other with syntenic group U5. These data confirm the chromosomal assignment of two syntenic groups and allow the assignment of a formerly unassigned syntenic group.

Y chromosome polymorphism in various breeds of cattle (Bos taurus) in Switzerland.

The evolutionary development of mammals involves mutations and fixations of chromosomal types. The Y chromosome polymorphism in cattle is important for the breeding strategy, since chromosomal incompatibilities in crossings result in fertility problems. In bulls of various breeds in Switzerland, data on chromosome status have been collected for over 20 years. Data from 7 years were analysed in this study through chromosome measurements and their normalization. Some highly significant differences were found between the 7 groups of breeds, especially between Holsteins and the original Swiss breeds Braunvieh and Simmental. Fleckvieh (purebred or crossbred) did not differ significantly from Black or Red Holsteins. The results were discussed with respect to fertility problems. The observed Y chromosome polymorphism should be taken into account in breeding, and research in this field should be continued.

Congenital progressive ataxia and spastic paresis, a hereditary disease in swine, maps to Chromosome 3 by linkage analysis

The congenital progressive ataxia (CPA) and spastic paresis in pigs, recently identified in Switzerland, is a disease with unknown etiology. A disease similar to CPA was described earlier by Rimaila-Pärnänen (1982). The affected animals show a neuropathic disorder, which occurs in piglets of both sexes within the first week after birth. The disease manifests itself within 1 or 2 days as a severe neuropathy, characterized by spastic gait, incoordination, and rapidly progressive ataxia in the hind limbs. Finally, the piglets remain lying down and are no longer able to support themselves. Histological examination of the central nervous system revealed no deficiency of stainable myelin nor any significant morphological changes. The disease was first observed in two litters of pigs (Table 1, matings 1 and 2), derived from two dams and one sire, all of Large White origin. The dams were cousins and not related to the sire, referring to the last two generations. Of the 23 offspring, seven (30.4%) were found to be affected and 16 (69.6%) were normal. The observed ratio of approximately 3:1 suggested that the disease may be controlled by a recessive allele. Therefore, affected animals were considered homozygous for the recessive allele ( cpa/cpa), and normal animals either heterozygous for the recessive allele ( CPA/cpa), or homozygous ( CPA/CPA). The present studies were conducted to: (1) confirm the autosomal recessive inheritance of CPA; and (2) map the CPA phenotype to the porcine genome. Of the 16 phenotypically normal animals, two males were mated to five females, and each female produced two litters (Table 1, mating 3–12). Of the 107 descendants, 17 animals (15.9%) showed ataxia and paresis syndromes. As their condition progressively worsened, the piglets were euthanized. Their average life span was (mean ± SD: 8.7 ± 8.3; n 4 17) days. In addition to the above offspring, nine piglets were produced from an unrelated family (Table 1, mating 13), of which three (33.3%) showed characteristics of the disease. To map the CPA phenotype, two to three highly informative microsatellite markers for each chromosome spread at intervals of about 40 cM were selected (Rohrer et al. 1996). According to conditions described in the original references (Rohrer et al. 1996), polymerase chain reaction (PCR) was carried out in a reaction volume of 25ml containing 100hg porcine genomic DNA extracted from blood, 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl2, 200 mM of each deoxynucleotide, 0.4 mM of forward and reverse primers, and 1.25 U of Taq DNA Polymerase (Pharmacia Biotech, Uppsala, Switzerland). PCR reactions (25 ml) were incubated for 30 cycles of 95°C for 30 s, 56°–62°C for 30 s, and 72°C for 30 s. With PrismTM Genescan-500 Rox marker (Applied Biosystems, Perkin-Elmer Corp., Foster City, Calif.), formamide diluted samples were analyzed on a 373A ABI Sequencer (Applied Biosystems Inc.). Linkage analysis was carried out with the CRIMAP program, version 2.4 (Green et al. 1990). In the inbreeding scheme among the phenotypically normal individuals, eight of the 12 litters produced atactic offspring (Table 1, matings No. 1, 2, 3, 6, 7, 8, 10, 12). Of the 95 descendants, 24 (25.3%) were found to be affected, while the remaining 71 (74.7%) were unaffected. The affected animals were classified as homozygous recessive ( cpa/cpa), and the unaffected as heterozygous (CPA/cpa) or homozygous ( CPA/CPA). Their parents were either heterozygous ( CPA/cpa), or homozygous ( CPA/CPA). Thex-test, calculated from the segregation data, showed that the observed ratios of thecpavs CPAalleles did not deviate significantly from the expected 1:3 ratio ( x 4 0.01; 0.9 <P < 0.95; 1 df). The genome scan revealed that the Sw902 allele (1894 size in bp), located on pig ( Sus scrofa ) Chromosome 3 (SSC3), co-segregated 100% with the recessive allele involved in the disease, while theSw902, Sw902 or Sw902 alleles cosegregated 100% with the normal allele (Table 1). Therefore, six additional markers in close proximity to marker Sw902were selected for further genotyping to generate a multipoint map covering the CPA region. Pairwise lod scores and recombination fractions forCPAand the seven marker loci are presented in Table 2. High lod scores of 16.9 and 11.6 were obtained for linkage of CPA with markersSw902and Sw1066respectively, the two markers exceeding a lod score of 47. Recombination was estimated to be 0.05 betweenSw1066andCPA,while no recombination occurred betweenSw902andCPA. It was computationally not feasible to perform a multipoint linkage analysis considering all eight loci jointly with n!/2 possible locus orders. Thus, the order Sw2618–Sw902–ACTG2–S0216 was fixed according to the genetic map of Rohrer et al. (1996), and the loci Sw460andSw1066were inserted sequentially with the CRIMAP “build” option. The most likely orderSw2618–Sw902– Sw1066–Sw460–ACTG2–S0216 fitted the data best, in accordance with Rohrer et al. (1996). The likelihood of six other loci orders did not differ by more than a factor of 1000, and they were, therefore, not considered significantly different. Similar results were obtained when other loci were assumed to be in a fixed order, and subsequently two additional loci were inserted. The marker order described by Rohrer et al. (1996), that is, Sw2618–S0094– Sw902–Sw1066–Sw460–ACTG2–S0216, was never rejected by our data. Therefore, this order was used in subsequent analyses. As expected, the estimated genetic distances and recombination rates are not completely in accordance with the data of Rohrer et al. (1996) (Fig. 1), probably owing to the different family material and limited number of meioses. The two orders ofCPA in adjacent intervals toSw902fit the Correspondence to: P. Vögeli Mammalian Genome 10, 1036–1038 (1999).

Molecular Genetics as a Diagnostic Tool in Farm Animals

In this review, the importance of molecular genetics for diagnostic applications in animal production and breeding is underlined. Recently, several new techniques and methods based on gene technology have been developed, such as the polymerase chain reaction, fluorescence in situ hybridization, and the use of microsatellite polymorphism. The examples include detection of favourable alleles of genes coding for milk proteins, recognition of negative recessive alleles in hereditary syndromes, the use of microsatellite variants for breeding purposes and parentage control, and application of specific DNA-probes for identification of Y-chromosome-bearing spermatozoa and the sex of embryos. It is to be understood that this list is not complete and more applications will undoubtedly show up in the future. For this review, the authors have mainly selected areas where they themselves or their co-workers have gained experience.

The bovine glutamine synthase gene (GLUL) maps to 10q33 and a pseudogene (GLULP) to 16q21

Species:Mouse Locus name: methionine synthase or 5-methyltetrahydrofolatehomocysteine methyltransferase Locus symbol:Mtr Map position: proximal–D13Mit1–1.06 cM ± 1.06 SE– Mtr, D13Bir4, D13Bir6–1.06 ± 1.06–D13Abb1e–2.13 ± 1.49–D13Bir7–distal Method of mapping:Mtr was localized by RFLP analysis of 96 animals from an interspecific backcross panel ((C57BL/6JEi × SPRET/Ei)F1 × SPRET/Ei) provided by The Jackson Laboratory, Bar Harbor, Me. (BSS panel) [1]. Database deposit information: The data are available from the Mouse Genome Database, accession number MGD-JNUM-39061. Molecular reagents:A 1095-bp mouse cDNA was obtained by reverse transcription/PCR of mouse liver RNA, with degenerate oligonucleotides based on regions of homology within the methionine synthase sequences of lower organisms. The two primers (D1730 and D1733), as described by Leclerc et al. [2], were successful in amplifying both human and mouse cDNAs. The PCR products from both species were subcloned and sequenced; they showed 89% identity. The mouse cDNA was labeled by random priming and hybridized to Southern blots of EcoRI-digested mouse genomic DNA. Allele detection:Allele detection was performed by RFLP analysis of an EcoRI polymorphism. The C57BL/6J strain has alleles of approximately 13 kb, while theMus spretusstrain has alleles of approximately 9 kb and 4 kb. A constant band of approximately 0.5 kb was seen in both strains. Previously identified homologs: Human MTR has been mapped to chromosomal band 1q43 by fluorescence in situ hybridization [2–4]. Discussion: Methionine synthase (EC 2.1.1.13, 5-methyltetrahydrofolate-homocysteine methyltransferase) catalyzes homocysteine remethylation to methionine, with 5-methyltetrahydrofolate as the methyl donor and methylcobalamin as a cofactor. Nutritional deficiencies and genetic defects in homocysteine metabolism result in varying degrees of hyperhomocysteinemia. Dramatic elevations in plasma and urinary homocysteine levels are associated with the inborn error of metabolism, homocystinuria. Consequent to the recent isolation of the human cDNA for methionine synthase [2–4], two groups of investigators have identified mutations in methionine synthase in homocystinuric patients [2, 5]. Mild elevations in plasma homocysteine are thought to be a risk factor for both vascular disease and neural tube defects [6–8]. A genetic variant in methylenetetrahydrofolate reductase (MTHFR), the enzyme that synthesizes 5-methyltetrahydrofolate for the methioninesynthase reaction, is the most common genetic determinant of hyperhomocysteinemia identified thus far [9]. Mild defects in the methionine synthase reaction are also potential candidates for hyperhomocysteinemia and the associated multifactorial diseases. A common variant has been reported for the human methionine synthase gene, but its physiologic consequences have not yet been determined [2, 4]. The mapping of the human MTR gene to 1q43 and of the mouse gene to proximal Chromosome (Chr) 13 is consistent with previous findings of human/mouse homologies between these 2 chromosomal regions; the human and mouse nidogen genes have been mapped to 1q43 and proximal Chr 13, respectively [10]. Several genes have already been implicated in neural tube defects in mice [11]. Studies involving the mouse methionine synthase gene will be useful in assessing the role of this important enzyme in the development of birth defects and/or vascular disease.