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Human cystathionine beta-synthase (CBS; EC 4.2.1.22) deficiency results in a recessive genetic disorder whose clinical and biochemical manifestations vary greatly among affected individuals. In an effort to identify and analyze mutations in the human CBS gene, we have developed a yeast expression system for human CBS. We have cloned and sequenced a human cDNA that codes for CBS and have expressed the human CBS protein in yeast cells lacking endogenous CBS. The human enzyme produced in yeast is functional both in vitro and in vivo. We have also cloned and sequenced the yeast gene, CYS4, that codes for CBS. The predicted human and yeast CBS proteins are 38% identical and 72% similar to each other, as well as sharing significant similarity with bacterial cysteine synthase. These results demonstrate the evolutionary conservation of CBS and establish the utility of a yeast expression system for studying human CBS.

Identification and recovery of transcribed sequences from cloned mammalian genomic DNA remains an important problem in isolating genes on the basis of their chromosomal location. We have developed a strategy that facilitates the recovery of exons from random pieces of cloned genomic DNA. The basis of this "exon trapping" strategy is that, during a retroviral life cycle, genomic sequences of nonviral origin are correctly spliced and may be recovered as a cDNA copy of the introduced segment. By using this genetic assay for cis-acting sequences required for RNA splicing, we have screened approximately 20 kilobase pairs of cloned genomic DNA and have recovered all four predicted exons.

Denaturing gradient gel electrophoresis (DGGE) can be used to distinguish two DNA molecules that differ by as little as a single-base substitution. This method detects approximately 50% of all possible single-base changes in DNA fragments ranging from 50 to approximately 1000 base pairs. To increase the number of single-base changes that can be distinguished by DGGE, we used the polymerase chain reaction to attach a 40-base-pair G + C-rich sequence, designated a GC-clamp, to one end of amplified DNA fragments that encompass regions of the mouse and human beta-globin genes. We show that this GC-clamp allows the detection of mutations, including the hemoglobin sickle (HbS) and hemoglobin C (HbC) mutations within the human beta-globin gene, that were previously indistinguishable by DGGE. In addition to providing an easy way to attach a GC-clamp to genomic DNA fragments, the polymerase chain reaction technique greatly increases the sensitivity of DGGE. With this approach, DNA fragments derived from less than 5 ng of human genomic DNA can be detected by ethidium bromide staining of the gel, obviating the need for radioactive probes. These improvements extend the applicability of DGGE for the detection of polymorphisms and mutations in genomic and cloned DNA.

By using 12 hamster-mouse hybrids segregating a mouse T(16;17)Bnr Robertsonian translocation chromosome in conjunction with 10 similar hybrids segregating normal mouse chromosomes, we have shown that the loci that control cellular sensitivity to interferon (IfRec) and code for the soluble enzyme superoxide dismutase (SOD-1) (superoxide:superoxide oxidoreductase; EC 1.15.1.1) are syntenic in the mouse and map to mouse chromosome 16. IfRec and SOD-1 are also syntenic in man. They have previously been assigned to the distal segment of the long arm of human chromosome 21, trisomy for which causes Down syndrome. Because both IfRec and SOD-1 map to mouse chromosome 16, it will now be possible to use mice trisomic for this chromosome to determine whether certain aspects of the Down syndrome phenotype in man are caused by an altered dosage of IfRec and SOD-1.

A theory is developed to predict marker retention and conditional retention or loss in radiation hybrids. Applied to multiple pairwise analysis of a human chromosome 21 data set, this theory fits much better than proposed alternatives and gives a physical map consistent with other evidence and robust with respect to errors to typing. Radiation hybrids have great promise to provide order and physical location at two levels of resolution, spanning the techniques of linkage and restriction fragments and not limited to polymorphic loci.

We have assigned the gene (B2m) coding for murine beta 2-microglobulin (B2M) to mouse chromosome 2 by using a novel panel of Chinese hamster-mouse somatic cell hybrid clones. Because of 35 independent primary hybrids used in this study were derived from two types of feral mice, each with a different combination of Robertsonian translocation chromosomes, as well as from mice with a normal complement of acrocentric chromosomes, analysis of 16 selected mouse enzyme markers provided data on the segregation of all 20 mouse chromosomes in these hybrids. Mouse B2M was identified in cell hybrids by immunoprecipitation with a species-specific anti-mouse B2M antiserum followed by two-dimensional polyacrylamide gel electrophoresis of the immunoprecipitated polypeptides. Enzyme analysis of the segregant clones excluded all chromosomes for B2m assignment except mouse chromosome 2, and karyotype analysis of nine informative hybrid clones confirmed the assignment of B2m to this chromosome. These results demonstrate that, in the mouse, as in man, B2m is not linked to the major histocompatibility or immunoglobulin loci.

Genetic linkage studies have mapped the Huntington disease (HD) mutation to the distal region of the short arm of human chromosome 4. Analysis of recombination events in this region has produced contradictory locations for HD. One possible location is in the region distal to the D4S90 marker, which is located within 300 kilobases of the telomere. Other crossover events predict a more centromeric position for HD. Here we analyze the telomeric region of 4p in detail. Cloned DNA segments were derived from this region by utilizing a radiation-induced somatic cell hybrid as a source of DNA combined with preparative pulsed-field gel electrophoresis to enrich for the telomeric fraction. Additional DNA was obtained by using the cloned segments as multiple start points for cosmid walks. This strategy proved to be an effective method for cloning 250 kilobases of DNA in the region telomeric to D4S90. Hybridization analysis with the cloned DNA did not provide any evidence for the presence of rearrangements of 100 base pairs or greater in the DNA of individuals affected with HD. We also found no change in the size or structure of the 4p telomere in these samples.

Aneuploid mouse embryos and fetuses are an important system for investigating the pathogenesis of the developmental and functional consequences of chromosome imbalance in mammals. However, the fact that almost all mouse aneuploids die in embryonic or fetal life restricts their usefulness for studies of loci and functions that are expressed only after birth. As an approach to rescuing aneuploid cells, we have prepared chimeras by aggregating aneuploid embryos with diploid embryos at the 8-16 cell stage. This technique has allowed us to produce trisomy 17 reversible diploid (Ts17 reversible 2n) chimeras containing cells of a trisomic state, which is ordinarily lethal at 10-12 days of gestation. All of the analyzed organs from the chimeras, including brain, liver, kidney, lung, muscle, heart, thymus, spleen, bone marrow, blood, and skin, contained both Ts17 and 2n cells. The proportion of Ts17 cells in each organ as estimated from coat color, enzyme markers (glucosephosphate isomerase), and karyotypes ranged from 5% to 85%, most commonly 20-40%. This is in contrast to the control 2n reversible 2n chimeras in which the 2n component on the same genetic background as the Ts17 generally comprised 60-90% of the cell population. The growth rates of the living Ts17 reversible 2n chimeras were in the lower half of the normal range, and the oldest animals are now age 14 mo. No progeny were obtained from Ts17 germ cells in the two fertile T217 reversible 2n chimeras. In comparison with analogous human situations, it is striking that, except for a kink in the tail of one living animal, none of the Ts17 reversible 2n chimeras had any discernible structural abnormalities. Our findings indicate that it is feasible to rescue cells from autosomal aneuploidies that otherwise result in early fetal death.