A new model for disruption of the ornithine decarboxylase gene, SPE1, in Saccharomyces cerevisiae exhibits growth arrest and genetic instability at the MAT locus.

Ornithine decarboxylase (ODC) is a rate-determining enzyme of the polyamine-biosynthetic pathway. We sought to produce cells with impaired ODC function in order to study the biological functions of polyamines. Saccharomyces cerevisiae strains were obtained by one-step gene replacement of a 900 bp fragment of the yeast ODC gene (SPE1) with the yeast URA3 gene. Spores derived from SPE1/spe1 cells germinated at reduced efficiency relative to SPE1/SPE1. Sustained growth of spe1 haploid mutants in polyamine-free medium led to intracellular polyamine depletion, reduction in budding index, G1 arrest and cessation of growth, and cells that were large and misshapen. All of these effects were completely reversed by adding polyamines to the medium, even after 5 days of polyamine starvation. A diploid yeast strain bearing two copies of disrupted spe1 lost heterozygosity at the mating-type locus more often when grown in the absence of polyamines than when grown in their presence, indicating that polyamine deficiency leads to either chromosome loss or to mitotic recombination.

Deletion of chromosome arm 17p DNA sequences in pediatric high-grade and juvenile pilocytic astrocytomas.

In adults, loss of heterozygosity for DNA on 17p has been shown in high-grade anaplastic astrocytomas (AAs) and glioblastomas multiforme (GMs), and mutation of the TP53 tumor suppressor gene has been reported in all grades of astrocytomas. Little is known, however, about 17p deletion and TP53 mutation in juvenile pilocytic astrocytomas (JPAs), the most common low-grade tumors seen in children. To elucidate the genetic characteristics of pediatric high-grade astrocytomas and JPAs, we performed restriction fragment length polymorphism analysis with probes derived from 17p and TP53 mutational studies in 28 tumor specimens. Telomeric chromosome arm 17p markers 144-D6 and ABR were lost in 6 (75%) of 8 informative tumors classified as high-grade (7 AAs, 1 GM) and in 2 (10%) of 20 informative JPAs. Loss of 17p probes centromeric to the TP53 gene were also detected in 3 AAs and 5 JPAs. Four of the 6 (66%) JPAs with losses of 17p DNA sequences recurred rapidly despite aggressive therapy, whereas only 5 of the other 14 (36%) recurred. Mutation of the TP53 gene was detected by polymerase chain reaction and denaturing gradient gel electrophoresis in only 1 JPA and 1 AA. These tumors were also examined for MDM2 gene amplification as an alternate inactivation mechanism for TP53 gene function: no instances of alteration were identified. These results suggest that a gene or genes in addition to TP53 on 17p may be involved in the etiology or progression of high-grade astrocytomas and aggressive JPAs in children.(ABSTRACT TRUNCATED AT 250 WORDS)

Physical mapping of chromosome 17p13.3 in the region of a putative tumor suppressor gene important in medulloblastoma.

Deletion mapping of a medulloblastoma tumor panel revealed loss of distal chromosome 17p13.3 sequences in tumors from 14 of 32 patients (44%). Of the 14 tumors showing loss of heterozygosity by restriction fragment length polymorphism analysis, 14 of 14 (100%) displayed loss of the telomeric marker p144-D6 (D17S34), while a probe for the ABR gene on 17p13.3 was lost in 7 of 8 (88%) informative cases. Using pulsed-field gel electrophoresis, we localized the polymorphic marker (VNTR-A) of the ABR gene locus to within 220 kb of the p144-D6 locus. A cosmid contig constructed in this region was used to demonstrate by fluorescence in situ hybridization that the ABR gene is oriented transcriptionally 5' to 3' toward the telomere. This report provides new physical mapping data for the ABR gene, which has not been previously shown to be deleted in medulloblastoma. These results provide further evidence for the existence of a second tumor suppressor gene distinct from p53 on distal chromosome 17p.

Diffuse leptomeningeal involvement by a ganglioglioma in a child. Case report.

Gangliogliomas are tumors composed of neuronal and glial elements that typically grow slowly by expansion only. This report describes a 20-month-old girl with a ganglioglioma that extensively involved the subarachnoid space; microscopic foci of tumor were found in the brain and spinal cord. Despite chemotherapy and radiation therapy, the child died 5 months after diagnosis. Molecular genetic analysis showed loss of chromosome 17p DNA sequences in the tumor tissue.

Involvement of multiple chromosome 17p loci in medulloblastoma tumorigenesis.

Loss of heterozygosity for sequences located on chromosome 17p in several tumor types is often associated with mutations in the tumor suppressor gene p53. We previously showed consistent deletion of chromosome 17p12-13.1 in medulloblastoma, a common childhood brain tumor. Using denaturing gradient gel electrophoresis and direct sequencing, we have detected p53 mutations in only two of 20 medulloblastoma specimens. Moreover, additional RFLP studies of these 20 specimens showed loss of heterozygosity at a more distal and distinct site, 17p13.3. Deletion of 17p almost invariably signified a negative prognosis. Our results suggest that p53 mutations may contribute to the pathogenesis of medulloblastoma in relatively few cases. The consistent deletion of other discrete loci on 17p suggests that additional or alternative tumor suppressor genes may contribute to the tumor's phenotype.

Identification of a germ-line mutation in the p53 gene in a patient with an intracranial ependymoma.

We detected a germ-line mutation of the p53 gene in a patient with a malignant ependymoma of the posterior fossa. This mutation, which was found at codon 242, resulted in an amino acid substitution in a highly conserved site of exon 7 of the p53 gene; the same mutation was found in both the germ-line and the tumor tissue. This is the most common region of previously described somatic p53 mutations in tumor specimens and of the germ-line p53 mutations in patients with the Li-Fraumeni cancer syndrome. Evaluation of the patient's family revealed several direct maternal and paternal relatives who had died at a young age from different types of cancer. The association of a germ-line p53 mutation with an intracranial malignancy and a strong family history of cancer suggests that p53 gene mutations predispose a person to malignancy and, like retinoblastoma mutations, may be inherited.

Loss of heterozygosity for chromosome 22 DNA sequences in human meningioma.

Monosomy of chromosome 22 in meningioma was the first consistent cytogenetic anomaly reported for a solid tumor. Although most meningiomas are isolated sporadic lesions, multiple and familial occurrences have been reported, usually in cases of documented neurofibromatosis 2 (NF2). Previous reports have placed the NF2 locus on chromosome 22, flanked by the markers D22S1 and D22S28. We report a restriction fragment-length polymorphism study of 16 meningiomas conducted using chromosome 22 probes. None of the patients had clinical findings or a family history of NF2, although two of them eventually developed multiple intracranial meningiomas. Detectable loss of chromosome 22 sequences was observed in 50% of informative patients. Deletion mapping of tumors with preserved sequences showed that the loss of chromosome 22 DNA overlapped the region previously linked to NF2, but also included a sequence distal to the NF2 locus. These results suggest that the oncogenesis of human meningioma involves inactivation of a chromosome 22 locus that may be in close proximity to the gene for NF2.

Deletion mapping of the medulloblastoma locus on chromosome 17p.

Isochromosome 17q has previously been observed consistently in cytogenetic studies of medulloblastoma, the most common posterior fossa neoplasm in children. We performed a restriction fragment length polymorphism (RFLP) investigation of medulloblastoma which showed a loss of chromosome 17p sequences in 45% of these tumors. This finding was predictive of a poor clinical response to treatment. A contiguous panel of markers permitted mapping of the deletion to 17p12-p13.1, the same chromosomal region for which loss of alleles has been shown in tumor specimens from patients with colon cancer, and the same region to which the p53 gene has been mapped. This suggests that medulloblastoma is associated with a recessive oncogene on chromosome 17p that may be involved in the genesis of several embryologically unrelated neoplasms and that the absence of this gene in tumor tissue has prognostic significance.

Cystic fibrosis: diagnostic testing and the search for the gene.

Cystic fibrosis (CF) is a lethal genetic disorder inherited as an autosomal recessive at a frequency of about 1/2000 in Caucasian populations. A DNA marker genetically linked to CF was identified through a collaborative effort by random screening with a collection of RFLP markers on a set of CF families. The marker (CRI-L917) was mapped to chromosome 7. Construction of a genetic linkage map spanning the entire chromosome has led to the identification of a subset of 11 markers close to and flanking the CF locus. Using techniques of pulsed-field gel electrophoresis, which allow very large DNA fragments to be separated, we used seven probes to generate a long-range restriction map covering 12 million base pairs surrounding the CF locus. Information from the map is being used to isolate new probes closer to the CF gene. Methods being developed will allow candidate genes to be tested for their ability to correct defects in ion transport in cultured CF cells.

A 12 megabase restriction map at the cystic fibrosis locus.

We have constructed a physical map of the chromosomal region containing the cystic fibrosis locus using seven DNA markers and pulsed-field gel electrophoresis methods. The map includes cleavage sites for 8 rare-cutting restriction enzymes and spans over 12 megabases (Mb) of DNA, with one unlinked probe covering an additional 5 Mb. To our knowledge, this is the largest segment of human DNA which has been restriction-mapped to date. We can identify thirteen putative HTF islands spaced at intervals of 0.3-3.2 Mb. The region between loci D7S8 and MET, where the CF gene lies, includes 1.4-1.9 Mb of DNA.

The human "interferon-beta 2/hepatocyte stimulating factor/interleukin-6" gene: DNA polymorphism studies and localization to chromosome 7p21.

The human interferon-beta 2 gene (IFNB2) product is identical to that for the B-cell stimulation factor-2 (BSF-2), the hybridoma growth factor (HGF) ("interleukin-6"), and the hepatocyte stimulating factor (HSF). Proteins derived from this gene mediate the plasma protein response to tissue injury (acute-phase response) and regulate the growth and differentiation of both B and T cells. By using the enzymes MspI, BstNI, and BglI, three polymorphic systems were detected with probes for the IFNB2 gene. The MspI and BglI polymorphisms are likely to be due to base pair substitutions; the BstNI polymorphism was revealed by nine other enzymes and is likely to be due to DNA insertions within 1 kb of the 3' flanking region of the gene. This region is rich in AT dinucleotides, and slippage at DNA replication may generate the insertions of between 0.07 and 0.23 kb that were observed. The polymorphic MspI site also lies within the vicinity of the fifth exon. The BglI polymorphic site is likely to lie in 5' flanking DNA. The three polymorphisms are separate, and a variety of haplotypes was observed. A low level of linkage disequilibrium exists between the MspI and the BglI alleles. MspI and BstNI polymorphisms were observed in Caucasoids, CAR Pygmies, Zaire Pygmies, Melanesians, and Chinese but at differing frequencies, and not all alleles were present in all populations. The BglI polymorphism was observed in Caucasoids and Africans only. Linkage studies involving the IFNB2 gene and 27 other chromosome 7 markers have localized it to between D7S135 and D7S370 at 7p22-p21.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of adrenocorticotropin in the cortisol and aldosterone responses to angiotensin II in conscious dogs.

The role of ACTH in the cortisol and aldosterone responses to iv angiotensin II (AII) infusion, (5, 10, and 20 ng kg-1 min-1) in dogs was evaluated by examining the effect of AII infusion in conscious dogs pretreated with dexamethasone to suppress endogenous ACTH secretion. AII infusion in untreated dogs produced dose-related increases in plasma cortisol and aldosterone concentrations. The plasma ACTH concentration also increased. Dexamethasone treatment lowered the basal cortisol concentration from 1.7 +/- 0.1 to 0.7 +/- 0.1 micrograms/dl (P less than 0.05) and the ACTH concentration from 52 +/- 3 to 41 +/- 4 pg/ml (P less than 0.05), and abolished the cortisol response to all doses of AII, indicating that ACTH was necessary for the response. On the other hand, the basal aldosterone concentration was not significantly affected by dexamethasone, although the aldosterone response to the highest dose of AII was reduced. Additional experiments were performed to determine if the cortisol and aldosterone responses to AII (20 ng kg-1 min-1) in dexamethasone-treated dogs are restored if the ACTH concentration is maintained near control levels by iv infusion of synthetic alpha ACTH-(1-24) (0.3 ng kg-1 min-1). AII still failed to increase the plasma cortisol concentration in this group of dogs; however, the aldosterone response was fully restored. To evaluate the effect of elevated ACTH levels on the steroidogenic effects of AII, dogs were treated with dexamethasone and a higher dose of ACTH (0.4 ng kg-1 min-1). This dose of ACTH increased the plasma cortisol concentration from 1.7 +/- 0.1 to 3.5 +/- 0.8 micrograms/dl (P less than 0.05), but did not significantly affect the plasma aldosterone concentration. In the presence of constant elevated levels of ACTH, AII (10 and 20 ng kg-1 min-1) increased the plasma cortisol concentration in dexamethasone-treated dogs, although the response to the 10 ng kg-1 min-1 dose was smaller than the response in untreated dogs. Infusion of AII at 5 ng kg-1 min-1 did not increase the plasma cortisol concentration. In contrast, the increased plasma aldosterone produced by AII infusion in dexamethasone-treated dogs was not altered in the presence of elevated ACTH levels. Finally, AII infusion did not alter the clearance of cortisol. Collectively, these results demonstrate that an increase in plasma ACTH is necessary for the cortisol response to AII infusion.(ABSTRACT TRUNCATED AT 400 WORDS)

Genetic analysis of cystic fibrosis: linkage of DNA and classical markers in multiplex families.

Linkage of cystic fibrosis (CF) to DNA and classical markers was studied in 36 families of two or three generations with at least two living affected children. Among the 79 affected children, no recombinants were detected between the disease and the markers MET and pJ3.11, previously shown to be linked to CF. No linkage between the human trypsin gene family (which appears to include at least 10 members) and CF was found, although not all genes of the trypsin family have been screened yet. In one of the CF families, recombination between MET and pJ3.11 was detected in an unaffected sib. Data from our families suggest that the gene order of markers among chromosome 7q is: (7cen;p8.33)collagen(COL1A2);DOCR1-917;paraoxonase+ ++(PON);(MET-cf-J3.11);T-cell receptor beta chain (TCRB);qter. There was no evidence for (or against) either postzygotic selection or meiotic drive to explain the high frequency of CF in Caucasian populations.