Fa-Ten Kao

The gene coding for a sphingolipid activator protein, SAP-1, is on human chromosome 10

SummarySAP-1 is a sphingolipid activator protein found in human tissues required for the enzymatic hydrolysis of GM1 ganglioside and sulfatide. It appears to be missing in patients who have a genetic lipidosis resembling juvenile metachromatic leukodystrophy. Using rabbit antibodies against human SAP-1 it could be visualized in extracts from cultured human skin fibroblasts after sodium dodecylsulfate-polyacrylamide gel electrophoresis, followed by electroblotting to nitrocellulose membrane and immunochemical staining (Western blotting). A series of 23 human-Chinese hamster ovary cell hybrids containing different human chromosomes were examined. The parent Chinese hamster ovary cells did not have a reacting protein in the region of human SAP-1. Only in the eight hybrid clones containing human chromosome 10 was a reacting protein identified. Other chromosomes were excluded by this method. Therefore the gene for SAP-1 and the genetic mutation resulting in a fatal lipidosis are located on human chromosome 10.

Confirmation of assignment of the human α1-crystallin gene (CRYA1) to chromosome 21 with regional localization to q22.3

SummaryThe crystallins are highly conserved structural proteins universally found in the eye lens of all vertebrate species. In mammals, three immunologically distinct classes are present, α-, β-, and γ-crystallins, and each class represents a multigene family. The α-crystallin gene family consists of α1-crystallin (CRYA1) and α2-crystallin (CRYA2) genes (previously designated αA-and αB-crystallin, respectively), which show extensive sequence homology. We constructed a synthetic oligonucleotide probe of 25 bases corresponding to a specific region of the human α1-crystallin gene sequence. This 25-mer probe bears little sequence homology to human α2-crystallin gene and does not cross-hybridize to α2-crystallin sequences in Southern blot analysis. Using this unique synthetic probe, we have demonstrated the identity of the α1-crystallin gene in human genomic DNA. In addition, we have also confirmed its chromosomal location on human chromosome 21. Finally, we have regionally localized the gene to q22.3 by using both Southern blot analysis of a panel of cell hybrids containing different parts of human chromosome 21, and in situ hybridization to metaphase chromosomes. The use of synthetic oligonucleotide probes specific for individual genes should be useful in identifying and mapping members of multigene families.

Assignment of the gene coding for phosphoribosylglycineamide formyltransferase to human chromosome 14.

Purine-requiring Chinese hamster ovary cell auxotrophs of the complementation class ade− E were hybridized with various human cells, and hybrids were isolated under selective conditions in which the retention of the complementing gene on the human chromosome is necessary for survival. Synteny analysis in 72 primary and secondary hybrid clones using isozyme, karyotypic, and biochemical methods provides evidence for an assignment of the gene for phosphoribosylglycineamide formyltransferase (GART, EC 2.1.2.2), deficient in ade− E mutants, to human chromosome 14. The importance of this gene assignment to the development of hypotheses regarding the organization, structure, and regulation of genes involved in the same biosynthetic pathway in mammalian cells is discussed.

Regional localization of the gene coding for sphingolipid activator protein SAP-1 on human chromosome 10

Sphingolipid activator protein SAP-1 is required for the enzymatic hydrolysis of GMI ganglioside and sulfatide. The gene coding for SAP-1 was previously mapped to human chromosome 10 using monospecific antibodies prepared against SAP-1 in synteny analysis of somatic cell hybrids. In this study, we used a cDNA probe for SAP-1 and in situ hybridization to regionally localize theSAP1 gene to the long arm of chromosone 10, region q21–22. Additional mapping data using cell hybrids containing partial chromosome 10 and skin fibroblasts with trisomy 10p are consistent with the in situ hybridization mapping results.

Three-region specific microdissection libraries for the long arm of human chromosome 2, regions q33-q35, q31-q32, and q23-q24

Three region-specific libraries have been constructed from the long arm of human chromosome 2, including regions 2q33-35 (2Q2 library), 2q31-32 (2Q3) and 2q23-24 (2Q4). Chromosome microdissection and the Mbol linker-adaptor microcloning techniques were used in constructing these libraries. The libraries comprised hundreds of thousands of microclones in each library. Approximately half of the microclones in the library contained unique or low-copy number sequence inserts. The insert sizes ranged between 50 and 800 bp, with a mean of 130–190 bp. Southern blot analysis of individual unique sequence microclones showed that 70–94% of the microclones were derived from the dissected region. 31 unique sequence microclones from the 2Q2 library, 31 from 2Q3, and 30 from 2Q4, were analyzed for insert sizes, the hybridizing genomic HindIII fragment sizes, and cross-hybridization to rodent species. These libraries and the short insert microclones derived from the libraries should be useful for high resolution physical mapping, sequence-ready reagents for large scale genomic sequencing, and positional cloning of disease-related genes assigned to these regions, e.g. the recessive familial amyotrophic lateral sclerosis assigned to 2q33-q35, and a type I diabetes susceptibility gene to 2q31-q33.

Region-specific microdissection library and single-copy microclones for human chromosome 2p11–p13

We report the construction and characterization of a region-specific microdissection library for human chromosome 2p11–p13. This library (designated 2P4 library) is large, comprising 600,000 recombinant microclones. Thirty to 40% of the clones contain unique sequences. The insert sizes range from 100 to 800 bp, with a mean of 380 bp. A subset of the microclones was selected, based on their weak or no hybridization to total human DNA, for further analysis. Of 50 single-copy microclones analyzed, 35 clones (70%) were derived from human and are chromosome 2-specific. The insert sizes and the hybridizing genomic HindIII fragments of these clones were also determined. The 2P4 microdissection library and the single-copy microclones from the library are useful in preparing STS (sequence-tagged site) to isolate corresponding YAC (yeast artificial chromosome) or other clones with large inserts and for isolating region-specific cDNA clones as candidate genes for cloning disease-related genes assigned to this region.

Assignment of human gene encoding testis-specific lactate dehydrogenase C to chromosome 11, region p14.3-p15.5

The human gene coding for lactate dehydrogenase C (LDHC), a testis-specific isozyme, has been assigned to a refined region of chromosome 11, p14.3–p15.5, in which the lactate dehydrogenase A gene LDHA also resides, by using somatic cell hybrids and in situ chromosome hybridization. This assignment clearly indicates the close physical proximity of the LDHC and LDHA genes and supports the evolutionary closeness of these two isozymes.

Complete set of eleven region-specific microdissection libraries for human chromosome 2.

The construction and characterization of 11 region-specific libraries for the entire human chromosome 2 have been completed, including four libraries for the short arm and six libraries for the long arm, plus a library for the centromere region. These libraries were constructed using the chromosome microdissection and microcloning technology. Eight libraries have been described previously. This paper presents the final three libraries: 2q21–q22 (designated 2Q5 library), 2q11–q14 (2Q6), and 2p11.1–q11.1 (2CEN). The sizes of the dissected regions ranged between 20 and 30 Mb, with the centromere region of about 4 Mb. All these libraries are large, potentially comprising hundreds of thousands of recombinant microclones. Between 77% and 97% of the microclones were shown to derive from respective dissected regions. From 26 to 66 unique sequence microclones were isolated and characterized in detail for each library. The microclones have short inserts, ranging between 50 and 600 bp, with a mean of about 200 bp. The short inserts can be conveniently sequenced as STSs to provide high density probes for the dissected region. A plasmid sub-library containing at least 20,000 microclones, and usually more, has been prepared from each library and deposited to ATCC for general distribution. The libraries have been used effectively in constructing high resolution physical maps and for contig assembly, as well as in positional cloning of disease genes assigned to the dissected region. Comparing to other chromosomes with detailed mapping information and densely populated probes, chromosome 2 remains largely under-exploited. The availability of a complete set of region-specific libraries and unique sequence microclones from the libraries should provide valuable resources for genome analysis, high resolution physical mapping, region-specific cDNA isolation, and positional cloning for chromosome 2.

YAC contig mapping of six expressed sequences encoded by human chromosome 21

Six cDNA clones from human chromosome 21 have been mapped in a set of complete YAC contig spanning the entire chromosome 21q. The mapping positions between two STSs on the YAC contig and the NotI coordinates starting from the telomere of 21q were determined for the cDNA clones. The YAC contig mapping positions agree well with those using a comprehensive somatic cell hybrid mapping panel.

Construction and characterization of region-specific microdissection libraries and single-copy microclones for short arm of human chromosome 2

The short arm of human chromosome 2, comprising approximately 93 million bp, has been divided into four regions to construct region-specific microdissection libraries to facilitate physical mapping and gene cloning. These four regions include 2p23-p25 (designated 2P1), 2p21-p23 (2P2), 2p14-p16 (2P3), and 2p11-p13 (2P4). Together with three previously constructed microdissection libraries of 2P1, 2P2 and 2P4, a fourth library for the region 2p14-p16 (2P3) has been constructed and characterized to complete all four region-specific libraries for the entire 2p. The 2P3 library is very large, potentially comprising 1,000,000 recombinant microclones with insert sizes ranging between 50 and 800 bp and a mean of 250 bp. Approximately 40% of the microclones contain unique sequences. Of the 77 single-copy microclones analyzed, 66 clones (86%) hybridized to both human and chromosome 2 DNAs, indicating that they were derived from human and are chromosome 2 specific. The hybridizing HindIII genomic fragments for the 66 microclones have also been determined.