A high-resolution whole genome radiation hybrid map of human chromosome 17q22-q25.3 across the genes for GH and TK.

We have constructed a whole genome radiation hybrid (WG-RH) map across a region of human chromosome 17q, from growth hormone (GH) to thymidine kinase (TK). A panel of 128 WG-RH hybrid cell lines generated by X-irradiation and fusion has been tested for the retention of 39 sequence-tagged site (STS) markers by the polymerase chain reaction. This genome mapping technique has allowed the integration of existing VNTR and microsatellite markers with additional new markers and existing STS markers previously mapped to this region by other means. The WG-RH map includes eight expressed sequence tag (EST) and three anonymous markers developed for this study, together with 23 anonymous microsatellites and five existing ESTs. Analysis of these data resulted in a high-density comprehensive map across this region of the genome. A subset of these markers has been used to produce a framework map consisting of 20 loci ordered with odds greater than 1000:1. The markers are of sufficient density to build a YAC contig across this region based on marker content. We have developed sequence tags for both ends of a 2.1-Mb YAC and mapped these using the WG-RH panel, allowing a direct comparison of cRay6000 to physical distance.

The chicken beta 2-microglobulin gene is located on a non-major histocompatibility complex microchromosome: a small, G+C-rich gene with X and Y boxes in the promoter.

beta 2-Microglobulin is an essential subunit of major histocompatibility complex (Mhc) class I molecules, which present antigenic peptides to T lymphocytes. We sequenced a number of cDNAs and two genomic clones corresponding to chicken beta 2-microglobulin. The chicken beta 2-microglobulin gene has a similar genomic organization but smaller introns and higher G+C content than mammalian beta 2-microglobulin genes. The promoter region is particularly G+C-rich and contains, in addition to interferon regulatory elements, potential S/W, X, and Y boxes that were originally described for mammalian class II but not class I alpha or beta 2-microglobulin genes. There is a single chicken beta 2-microglobulin gene that has little polymorphism in the coding region. Restriction fragment length polymorphisms from Mhc homozygous lines, Mhc congenic lines, and backcross families, as well as in situ hybridization, show that the beta 2-microglobulin gene is located on a microchromosome different from the one that contains the chicken Mhc. We propose that the structural similarities between the beta 2-microglobulin and Mhc genes in the chicken are due to their presence on microchromosomes and suggest that these features and the microchromosomes appeared by deletion of DNA in the lineage leading to the birds.

The role of SOX9 in autosomal sex reversal and campomelic dysplasia.

In eutherian mammals, the Y-chromosome gene SRY is required for induction of testis development. Although the Y chromosome is sex determining, loci located elsewhere in the genome participate in the complex cascade of genetic interactions required to form a testis. Male to female sex reversal (46,XY females) occurs at a high frequency in individuals afflicted with the skeletal malformation syndrome campomelic dysplasia. Chromosomal translocations in individuals with both syndromes had localized an autosomal sex reversal locus (SRA1) and a campomelic dysplasia locus (CMPD1) to the long arm of human chromosome 17. The molecular cloning of a translocation breakpoint in a sex reversed campomelic dysplasia patient revealed its proximity to SOX9, a gene which is related to SRY. Analysis of SO X9 in patients without chromosomal rearrangements demonstrated single allele mutations in sex reversed campomelic individuals, linking this gene with both bone formation and control of testis development. Identification of SO X9 as SRA1/CMPD1 and the role of SO X9 mutations in sex reversal and campomelic dysplasia are discussed.

Mutations in SOX9, the gene responsible for Campomelic dysplasia and autosomal sex reversal.

Campomelic dysplasia (CD) is a skeletal malformation syndrome frequently accompanied by 46,XY sex reversal. A mutation-screening strategy using SSCP was employed to identify mutations in SOX9, the chromosome 17q24 gene responsible for CD and autosomal sex reversal in man. We have screened seven CD patients with no cytologically detectable chromosomal aberrations and two CD patients with chromosome 17 rearrangements for mutations in the entire open reading frame of SOX9. Five different mutations have been identified in six CD patients: two missense mutations in the SOX9 putative DNA binding domain (high mobility group, or HMG, box); three frameshift mutations and a splice-acceptor mutation. An identical frameshift mutation is found in two unrelated 46,XY patients, one exhibiting a male phenotype and the other displaying a female phenotype (XY sex reversal). All mutations found affect a single allele, which is consistent with a dominant mode of inheritance. No mutations were found in the SOX9 open reading frame of two patients with chromosome 17q rearrangements, suggesting that the translocations affect SOX9 expression. These findings are consistent with the hypothesis that CD results from haploinsufficiency of SOX9.

Campomelic dysplasia and autosomal sex reversal caused by mutations in an SRY-related gene.

Induction of testis development in mammals requires the presence of the Y-chromosome gene SRY. This gene must exert its effect by interacting with other genes in the sex-determination pathway. Cloning of a translocation chromosome breakpoint from a sex-reversed patient with campomelic dysplasia, followed by mutation analysis of an adjacent gene, indicates that SOX9, an SRY-related gene, is involved in both bone formation and control of testis development.

Mapping of the chicken N-CAM gene and a myosin heavy chain gene: avian microchromosomes are not genetically inert reserves of DNA.

Physical mapping of the chicken neural cell adhesion molecule gene and of a chicken myosin fast-white heavy chain gene via in situ hybridization has assigned both loci to different-sized microchromosomes in this species. Two genomic cloned chicken DNA probes of 16 kb (lambda cN6) and 15 kb (DCM 11), respectively, were biotin labeled for fluorescent detection of the genes. The implications derived from these results, together with further data just now beginning to accumulate regarding the genetic content of avian microchromosomes, confirm an unexpectedly active role for these minute chromosomes.

Sex-linkage of the chicken ornithine transcarbamylase gene.

The chicken ornithine transcarbamylase gene has been mapped to the short arm of the Z chromosome, near the centromere. Radioactive in situ hybridization was performed using a human cDNA clone, and chromosomes were scored for silver grain accumulation. This represents the first documented case of a mammalian X-linked gene also being sex-linked in an avian species. The significance of this finding in the context of sex chromosome evolution is briefly discussed. Further, differential sex-related OTC expression, whereby females (ZW) consistently produce higher levels of the enzyme than males (ZZ), must imply a peculiar mode of OTC transcription regulation.

In situ mapping of the chicken progesterone receptor gene and the ovalbumin gene.

The chicken progesterone receptor (cPR) gene and the ovalbumin (OA) gene, a target of cPR regulation, have been mapped via fluorescent in situ hybridization to the two largest chromosomes of the chicken karyotype. cPR is subtelomeric on the long arm of chromosome 1 and OA is on the long arm of chromosome 2, close to the centromere. A 35-kb cosmid probe for the cPR gene and two genomic fragments of 9.2 and 15 kb for the OA gene were biotin-labeled for nonradioactive localization of the two chicken loci.

Mapping the beta NGF gene in situ to a microchromosome in chicken.

The chicken nerve growth factor (beta NGF) gene has been mapped by fluorescent in situ hybridization to a pair of microchromosomes, confirming previous reports of the existence of a single gene locus. A 39-kb genomic fragment cloned in a cosmid vector and including the 5' end of the beta NGF locus was biotinylated for nonradioactive detection of the gene. This report adds to the increasing evidence proving microchromosomal localization of highly conserved and biologically fundamental genes. The implications of such genes belonging to very small linkage groups for the transmission of alleles from generation to generation together with the relevance of nonisotopic in situ hybridization for avian gene mapping are considered.

Microchromosomal assignment of the chicken ovotransferrin and adenylate kinase genes.

Ovotransferrin, an egg-white protein implicated in the transfer of trace elements from the hen oviduct to the developing avian embryo, and cytosolic adenylate kinase, an essential enzyme involved in the interconversion of adenine nucleotides in energetically active tissues, have been mapped to two separate chicken microchromosomes by fluorescent in situ hybridization. Considering present and previous data, the possibility of loss of intron material resulting in the compactation of genes in chicken microchromosomes is briefly discussed.

Assignment of the chicken tyrosine hydroxylase gene to chromosome 6 by FISH.

The gene for tyrosine hydroxylase, the first and rate-limiting enzyme in the biosynthetic pathway of catecholamine neurotransmitters, has been localized in situ to chromosome 6 in the chicken. It is the first DNA marker to be reported for this telocentric macrochromosome. Use of a 45-kb biotinylated chicken-specific cosmid probe and a sensitive fluorescent detection system proved to be highly efficient, with over 90% of metaphases showing positive hybridization signals on one or (usually) both chromosome 6 homologs, in physically mapping this single-gene locus.

Linkage of the chicken MHC to the nucleolus organizer region visualized using non-isotopic in situ hybridization.

Linkage of the chicken major histocompatibility complex (B-complex) to the nucleolus organizer regions (NOR) has been confirmed in situ. Nonradioactive hybridization to metaphase chromosomes using a pool of biotinylated cDNA probes for the B-F, B-L beta, and B-G genes of the B-complex gave positive signals on one or a pair of microchromosomes. Subsequent staining with silver nitrate proved these microchromosomes to be the NOR-bearing pair. The value of employing nonisotopic in situ hybridization techniques for mapping of genes and gene complexes, especially when dealing with microchromosomes, is stressed.

The dystrophin gene is autosomally located on a microchromosome in chicken.

The dystrophin gene has been mapped to a pair of microchromosomes in Gallus domesticus. In situ hybridization using a pool of biotinylated human cDNA probes allowed detection of this huge single-copy sequence without having to employ isotopic labeling. The autosomal nature of the DMD gene in chicken is supported by molecular data from quantitative Southern blot analysis and is in sharp contrast to that in all eutherian mammals studied, where it is a characteristically X-linked locus. With previous data taken into consideration, these results should prove significant in understanding the evolution of sex chromosomes during speciation as well as highlighting the importance of avian microchromosomes.

Immunocytogenetics. VI. A nonhistone antigen is cell type-specially associated with constitutive heterochromatin and reveals condensation centers in metaphase chromosomes.

We report a nonhistone antigen to be cell type-specifically associated with constitutive heterochromatin. Human autoantibodies were used to analyze by indirect immunofluorescence the pattern of association of the antigenic protein with the heterochromatin of murine chromosomes, as well as those of other representative vertebrate species. The evolutionary stability of its cell type-specific distribution pattern suggests that this nonhistone antigen plays an important role in the structure and/or function of constitutive heterochromatin. In mitotic chromosomes, the antigen was localized to discrete granules scattered throughout the entire chromatin. These structural elements may function as condensation centers, with each granule representing an aggregation of anchoring complexes for the chromatin loops.

Immunocytogenetics. IV. Human autoantibodies to heterochromatin-associated proteins.

We report here a novel class of human autoantibodies with immunological affinity for constitutive heterochromatin. Indirect immunofluorescence localized different proteinaceous antigens to the AT-rich paracentromeric heterochromatin of mouse chromosomes, the GC-rich heterochromatic regions of sheep chromosomes, and the large C-banded regions of human chromosomes 1, 9, and 16. Minor amounts of the heterochromatin-associated proteins were uniformly distributed on the chromosomes of all vertebrate species. Their antigenic determinants have been highly conserved during evolution. The chromosomal distribution of the heterochromatin-associated antigens is not altered in interspecific somatic cell hybrids but apparently reflects a stable structural property germane to each chromosome type. The antigenic proteins remain bound to their respective epitopes throughout the entire cell cycle and in meiosis. The heterochromatin-associated antigens represent a major nonhistone component of the mitotic chromosome scaffold and the interphase nuclear matrix. Immunoblotting performed with a human autoimmune serum to mouse heterochromatin revealed a characteristic pattern of polypeptides with molecular weights ranging from 35 to 120 kDal. Drugs that interfere in vivo with the higher-order chromatin structure had no effect on the expression and chromosomal distribution of the heterochromatin-associated antigens. However, the antibody binding sites in chromatin can be completely masked by treating fixed cell preparations with certain DNA-binding ligands.