Gail Bruns

Transposition and amplification of oncogene-related sequences in human neuroblastomas

We have cloned a 2.0-kb EcoRI fragment of human genomic DNA (NB-19-21) which has homology to the v-myc oncogene but is distinct from the classical c-myc gene. This sequence is amplified from 25- to 700-fold in eight of nine tested human neuroblastoma cell lines which contain either homogeneously staining regions or double minutes (HSRs or DMs), the caryological manifestations of amplified genes. In the remaining line, the c-myc proto-oncogene is amplified approximately 30-fold. NB-19-21 hybridizes to a 3.2-kb cytoplasmic, poly(A)+ RNA species that is abundant only in lines in which the sequence is amplified. We propose that the gene encoding the NB-19-21-related RNA species may represent a new oncogene, which we call N-myc. NB-19-21 derives from chromosome 2; but in the five HSR-containing lines that have amplified this sequence, none has HSRs on chromosome 2. NB-19-21 is associated with DMs in a DM-containing line. A second, randomly cloned, amplified DNA segment from the HSR of one of the neuroblastoma lines is amplified in a subset of the lines in which NB-19-21 is amplified. In addition, this probe identifies a novel joint in the amplification unit of one line relative to that of the others. We suggest that, in the eight lines which have amplified NB-19-21, the amplification units are overlapping, but not identical, and that transposition of the common sequences may occur prior to amplification.

Homozygosity of Chromosome 13 in Retinoblastoma

We studied the frequency of chromosome 13 homozygosity in tumor tissue obtained directly from eyes harboring retinoblastomas. The data indicate that approximately half of all retinoblastomas are homozygous for large portions of 13q, that the homozygosity occurs in vivo and not as an event secondary to culture of the tumor cells, that chromosome 13 homozygosity is not correlated with the degree of histopathologic differentiation of the tumor, and that the homozygosity occurs in both sporadic and hereditary retinoblastomas. The development of chromosome 13 homozygosity may represent a fundamental event in the oncogenesis of a considerable number of retinoblastomas. This finding may have implications for the genetic counseling of patients with hereditary retinoblastoma. It may also be important in understanding the mechanism of oncogenesis of other tumors, especially hereditary tumors.

The T cell receptor β chain genes are located on chromosome 6 in mice and chromosome 7 in humans

Homologous clones that encode the beta chain of the T cell antigen receptor have been isolated recently from both murine and human cDNA libraries. These cDNA clones have been used in connection with interspecies hybrid cell lines to determine that the murine T cell receptor gene is located on chromosome 6 and the human gene on chromosome 7. In situ hybridization confirms these data and further localizes these genes to band B of chromosome 6 in the mouse and bands 7p13-21 in the human genome. The organization of the T cell antigen receptor J beta gene segments and C beta genes appears to be conserved, since very few intraspecies polymorphisms of restriction fragment length have been detected in either mouse or human DNA.

The genomic organization and expression of the WT1 gene

The Wilms tumor gene WT1, a proposed tumor suppressor gene, has been identified based on its location within a homozygous deletion found in tumor tissue. The gene encodes a putative transcription factor containing a Cys/His zinc finger domain. The critical homozygous deletions, however, are rarely seen, suggesting that in many cases the gene may be inactivated by more subtle alterations. To facilitate the search for smaller deletions and point mutations we have established the genomic organization of the WT1 gene and have determined the sequence of all 10 exons and flanking intron DNA. The pattern of alternative splicing in two regions has been characterized in detail. These results will form the basis for future studies of mutant alleles at this locus.

Monoamine oxidase deficiency in males with an X chromosome deletion.

Mapping of the human MAOA gene to chromosomal region Xp21-p11 prompted our study of two affected males in a family previously reported to have Norrie disease resulting from a submicroscopic deletion in this chromosomal region. In this investigation we demonstrate in these cousins deletion of the MAOA gene, undetectable levels of MAO-A and MAO-B activities in their fibroblasts and platelets, respectively, loss of mRNA for MAO-A in fibroblasts, and substantial alterations in urinary catecholamine metabolites. The present study documents that a marked deficiency of MAO activity is compatible with life and that genes for MAO-A and MAO-B are near each other in this Xp chromosomal region. Some of the clinical features of these MAO deletion patients may help to identify X-linked MAO deficiency diseases in humans.

Human monoamine oxidase gene (MAOA): chromosome position (Xp21-p11) and DNA polymorphism.

An essentially full-length cDNA clone for the human enzyme monoamine oxidase type A (MAO-A) has been used to determine the chromosomal location of a gene encoding it. This enzyme is important in the degradative metabolism of biogenic amines throughout the body and is located in the outer mitochondrial membrane of many cell types. Southern blot analysis of PstI-digested human DNA revealed multiple fragments that hybridized to this probe. Using rodent-human somatic cell hybrids containing all or part of the human X chromosome, we have mapped these fragments to the region Xp21-p11. A restriction fragment length polymorphism (RFLP) for this MAOA gene was identified and used to evaluate linkage distances between this locus and several other loci on Xp. The MAOA locus lies between DXS14 and OTC, about 29 cM from the former.

The 35 kd pulmonary surfactant-associated protein is encoded on chromosome 10

SummaryThe genomic components identified by each of two closely related cDNA clones for the major 35 kilodalton non-serum surfactant-associated proteins (PSP-A) were shown to derive from human chromosome 10 by Southern blot analysis of DNAs from human-rodent somatic cell hybrids. By in situ hybridization to human metaphase chromosomes, the cDNA probes were localized to the region 10q21-q24.

Transformation associated p53 protein is encoded by a gene on human chromosome 17

The human gene for the transformation-associated p53 phosphoprotein (P53)was assigned to the short arm of chromosome 17 using human-rodent somatic cell hybrids and Southern filter hybridization of cell hybrid DNA. The filters were hybridized to radiolabeled DNA from a genomic clone which contained P53nucleotide sequences. Hybridization of the probe to a 2.5-kb human DNA fragment in HindIII-digested DNA was used to identify the human P53gene.

Localisation of genetic markers and orientation of the linkage group on chromosome 19

SummaryA panel of human-rodent somatic cell hybrids containing translocation derivatives of human chromosome 19 has been used to assign the markers peptidase D, complement component 3, lysosomal mannosidase, lysosomal DNAase, chorionic gonadotropin β-subunit, and a new polymorphic DNA sequence, to specific regions of chromosome 19. This has allowed the relative orientations of the genetic and physical maps to the established, and provides the framework for a search for the genes responsible for inherited disorders on chromosome 19, such as myotonic dystrophy and neurofibromatosis.

Genetic Mapping of the Wiskott-Aldrich Syndrome with Two Highly-Linked Polymorphic DNA Markers

The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive genetic disease in which the molecular defect is unknown. In 15 families with WAS, seven restriction fragment length polymorphic loci from the X chromosome were used to map the disease locus. Of the eight intervals studied, the likelihood of the WAS gene lying between DXS7 (Xp11.3) and DXS14 (Xp11) was at least 128 times higher than that for any other interval. The most likely gene order is DXS84-OTC-DXS7-WAS-DXS14-DXS1-PGK-DXYS1. Close genetic linkage to DXS7 and DXS14 permits accurate prenatal diagnosis and carrier detection with greater than 98% confidence in fully informative WAS families.