Karin Schmitt

Close linkage between bovine prolactin and BoLA-DRB3 genes: genetic mapping in cattle by single sperm typing.

The major histocompatibility complex and prolactin (PRL) genes are syntenic in humans and cattle but the genetic distance between these loci has not been determined for either species. In this study, the sperm typing technique was used to measure the recombination frequency between the bovine lymphocyte antigen (BoLA)-DRB3 and PRL loci. A total of 300 sperm were typed from one doubly heterozygous bull for segregation of DRB3 and PRL alleles. Sperm typing was performed using the polymerase chain reaction (PCR) and restriction enzyme cleavage of the PCR products, followed by resolution of the restriction fragments in polyacrylamide gels. Digestion with the restriction endonuclease RsaI allowed the unambiguous discrimination of alleles for both loci. The maximum likelihood estimation of the recombination fraction theta = 0.04, with a 95% confidence interval of 0.01 to 0.07. Close linkage between PRL and DRB3 has important implications for marker-assisted selection in animal breeding since PRL has been shown to be closely linked to a locus that affects milk yield, and BoLA loci influence susceptibility to a number of infectious diseases. Our results demonstrate the general applicability of the sperm typing procedure for gene mapping in species other than humans and provide an example of how parallel efforts to map the genomes of agriculturally important species of animals can have a positive impact on the development of a primary human linkage map.

Molecular analysis of 9p deletions associated with XY sex reversal: refining the localization of a sex-determining gene to the tip of the chromosome.

We thank Dr. C. P. Bennet, R. Hawkins, and Dr. O. Zuffardi, for providing us with some of the patient material; Giuliana Peverali, for technical support in the microsatellite typing; M. Davies, for technical support in tissue culture; and Drs. C. Farr and E.A. Oakenfull, for critical reading of the manuscript. This work was supported by Welcome grant 035201/Z/92 (to P.N.G and S.G.), EU grant Biomed2-CT960790 (to P.N.G.), a Telethon research fellowship (to S.G.), and an EMBO long-term fellowship (to K.S.).

Construction and characterization of zebrafish whole genome radiation hybrids.

Publisher Summary This chapter describes the construction and characterization of zebrafish whole genome radation hybrids. Zebrafish whole genome radiation hybrids are produced by fusing irradiated zebrafish cells to a hamster cell line that is hypoxanthine phosphoribosyl transferase (HPRT) or thymidine kinase (TK) deficient. The irradiation procedure randomly breaks the zebrafish genome, and the DNA fragments generated are rescued via the fusion process. Surviving cells are grown in hypoxanthine-aminopterinethymidine (HAT) medium, which selects for hybrids containing the zebrafish HPRT gene or TK gene. After picking the surviving hybrid clones and DNA extraction, the panel of hybrids is tested by polymerase chain reaction (PCR) for retention of genomic fragments by typing several microsatellite markers across the genome. For large-scale mapping or positional cloning projects, statistical programs are available to calculate map order and distances based on PCR typing of many markers across the hybrid panel. The advantages of whole genome radiation hybrids are (1) the ability to map any marker as long as it differs between rodent and zebrafish and (2) tailoring panels to different mapping resolutions depending on the application by adjusting the radiation dose.

Regional assignment of human ESTs by whole-genome radiation hybrid mapping.

The UK HGMP Resource Centre’s collection of human partial cDNA sequences (ESTs) have been examined for suitability for mapping by PCR on a panel of somatic cell hybrids. The chromosomal assignments of 92 ESTs were determined with a monochromosomal hybrid panel, and a subset of 45 were linked to genetic markers with a panel of whole-genome radiation hybrids (WG-RHs). These results demonstrate the potential of WG-RHs to construct a transcript map of the human genome.

Single‐Sperm Typing

This unit presents protocols for sperm isolation using two different methods, amplification of simple sequence‐length polymorphisms (SSLP) and/or single nucleotide polymorphisms (SNP) from single cells or whole genome‐amplified single cells using primer extension preamplification (PEP), and discusses the statistical analysis of sperm‐typing recombination data. Newer methods for studying recombination over very short distances (a few kilobases) using total sperm DNA and allele‐specific PCR are also discussed.