Pathogenesis of precocious puberty in hypothalamic hamartoma

Astroglial-derived factors, as transforming growth factor (TGF)alpha and TGFbeta, act in the hypothalamus to activate luteinizing hormone-releasing hormone (LHRH) secretion. Hypothalamic hamartomas (HHs) contain normal nervous tissue in a heterotopic location. When symptomatic, they cause precocious puberty and/or characteristic gelastic seizures. Thus far, the pathogenesis of these alterations remains unknown. By examining two HHs associated with sexual precocity, we found that they contained astroglial cells expressing TGFalpha, but no LHRH neurons. In a third patient with HH, only epilepsy was present, but precocious puberty developed shortly after surgery, probably as a consequence of a surgery-induced lesion. These results imply that some HHs induce sexual precocity by activating endogenous LHRH secretion via astroglial-derived factors.     

Minisatellite instability at the Adh locus reveals somatic polymorphism in amphioxus

Amphioxus (subphylum Cephalochordata) is the closest living relative to vertebrates and widely used for phylogenetic analyses of vertebrate gene evolution. Amphioxus genes are highly polymorphic, but the origin and nature of this variability is unknown. We have analyzed the alcohol dehydrogenase locus (Adh3) in two amphioxus species (Branchiostoma lanceolatum and Branchiostoma floridae) and found that genetic variation is related to repetitive DNA sequences, mainly minisatellites. Small pool-PCR assays indicated that allelic variants are generated by minisatellite instability. We conclude that the generation of new forms was not preferentially linked to germline processes but rather to somatic events leading to mosaic adult animals. Furthermore, most Adh minisatellites belong to a novel class, which we have named mirages. Their distinctive feature is that the repeat subunit spans the exon-intron boundaries and generates potential duplications of the splice sites. However, splicing may not be compromised as no aberrant mRNA variants were detected.     

Identification of genotype at the sex-determining locus in the honeybee

Genotype at the sex-determining locus in the honeybee, Apis mellifera, is identified by brood viability levels.     

Large-scale chromosomal restructuring is induced by the transposable element tam3 at the nivea locus of antirrhinum majus

The transposable element, Tam3, gives rise to large-scale (greater than 1 kb) chromosomal rearrangements at a low frequency, when it is inserted at the nivea locus of Antirrhinum majus. Although some deletions may result from imprecise excision of Tam3, rearrangements involving deletion, dispersion and inverted duplication of flanking sequences, where Tam3 remains in situ, have also been identified. These rearrangements have been mapped at the molecular level, and the behavior of Tam3 following rearrangement has been observed. It is clear that Tam3 has enormous potential to restructure chromosomes through successive rounds of large-scale rearrangements. The mechanisms by which such rearrangements might arise are discussed.     

A comparison of somatic chromosomal instability in tissue culture regenerants from Medicago media Pers

Two cultivars (Heinrichs, Reaver) and two breeding lines (Br1, Le1) from Medicago media were cultured in a media protocol consisting of a high concentration 2,4-D induction step. Regenerants were produced from all four stocks. Representative samples from each regenerant population along with the corresponding control population were cytologically analyzed for chromosomal and pollen abnormalities. While numerical changes in chromosome numbers were found in all regenerant populations, there was considerable variation between the four stock groups. Heteroploidy was observed for both hypo and hyper aneuploid regenerants, but there were no differences in pollen stainability between hypo and hyper aneuploid regenerants and euploid regenerants. Tissue culture regenerants generally produced a lower pollen stainability percent as compared to control populations grown from seeds. Gross and cryptic changes in chromosomes, or hormonal carry over effects or both were considered causes for poor pollen stainability in tissue culture regenerants. Cytological analyses indicate that the cultivar might play an important role in the cytological stability or instability of regenerant populations. Exploitation of this naturally existing situation to produce euploid regenerants for field experiments and to obtain gross cytological stability in somaclones is discussed.     

Identification and chromosomal mapping of a third mouse runt-like locus

The Drosophila runt gene, which controls early events in embryogenesis, has been shown to have homologues in human and mouse. The human gene on 21q22 is involved in the t(8;21) associated with acute myeloid leukemia. Two mouse runt-like loci encoding DNA-binding proteins have been identified. We report here the isolation and partial sequence of a molecular clone of a third mouse runt-like locus. By using a panel of somatic cell hybrids and interspecific backcross mice, we map the novel locus to the telomeric region of mouse chromosome 4.     

Transposon Ac/Ds-induced chromosomal rearrangements at the rice OsRLG5 locus

Previous studies have shown that pairs of closely-linked Ac/Ds transposable elements can induce various chromosomal rearrangements in plant genomes. To study chromosomal rearrangements in rice, we isolated a line (OsRLG5-161) that contains two inversely-oriented Ds insertions in OsRLG5 (Oryza sativa Receptor like kinase Gene 5). Among approximately 300 plants regenerated from OsRLG5-161 heterozygous seeds, 107 contained rearrangements including deletions, duplications and inversions of various sizes. Most rearrangements were induced by previously identified alternative transposition mechanism. Furthermore, we also detected a new class of rearrangements that contain juxtaposed inversions and deletions on the same chromosome. We propose that these novel alleles were generated by a previously unreported type of alternative transposition reactions involving the 5' and 3' termini of two inversely-oriented Ds elements located on the same chromatid. Finally, 11% of rearrangements contained inversions resulting from homologous recombination between the two inverted Ds elements in OsRLG5-161. The high frequency inheritance and great variety of rearrangements obtained suggests that the rice regeneration system results in a burst of transposition activity and a relaxation of the controls which normally limit the transposition competence of individual Ds termini. Together, these results demonstrate a greatly enlarged potential of the Ac/Ds system for plant chromosome engineering.     

Identification of the human chromosomal region containing the iridogoniodysgenesis anomaly locus by genomic-mismatch scanning.

Genome-mismatch scanning (GMS) is a new method of linkage analysis that rapidly isolates regions of identity between two genomes. DNA molecules from regions of identity by descent from two relatives are isolated based on their ability to form extended mismatch-free heteroduplexes. We have applied this rapid technology to identify the chromosomal region shared by two fifth-degree cousins with autosomal dominant iridogoniodysgenesis anomaly (IGDA), a rare ocular neurocristopathy. Markers on the short arm of human chromosome 6p were recovered, consistent with the results of conventional linkage analysis conducted in parallel, indicating linkage of IGDA to 6p25. Control markers tested on a second human chromosome were not recovered. A GMS error rate of approximately 11% was observed, well within an acceptable range for a rapid, first screening approach, especially since GMS results would be confirmed by family analysis with selected markers from the putative region of identity by descent. These results demonstrate not only the value of this technique in the rapid mapping of human genetic traits, but the first application of GMS to a multicellular organism.     

Identification of a novel T-insertion polymorphism at the DMD locus

We have identified a novel T-insertion polymorphism located in the second intron of the dystrophin gene. This polymorphism should prove useful in linkage studies in Duchenne and Becker muscular dystrophy families in addition to the previously described markers.     

Chromosome abnormalities in sperm of individuals with constitutional sex chromosomal abnormalities

The most common type of karyotype abnormality detected in infertile subjects is represented by Klinefelter's syndrome, and the most frequent non-chromosomal alteration is represented by Y chromosome long arm microdeletions. Here we report our experience and a review of the literature on sperm sex chromosome aneuploidies in these two conditions. Non mosaic 47,XXY Klinefelter patients (12 subjects) show a significantly lower percentage of normal Y-bearing sperm and slightly higher percentage of normal X-bearing sperm. Consistent with the hypothesis that 47,XXY germ cells may undergo and complete meiosis, aneuploidy rate for XX- and XY-disomies is also increased with respect to controls, whereas the percentage of YY-disomies is normal. Aneuploidy rates in men with mosaic 47,XXY/46,XY (11 subjects) are lower than those observed in men with non-mosaic Klinefelter's syndrome, and only the frequency of XY-disomic sperm is significantly higher with respect to controls. Although the great majority of children born by intracytoplasmic sperm injection from Klinefelter subjects are chromosomally normal, the risk of producing offspring with chromosome aneuploidies is significant. Men with Y chromosome microdeletions (14 subjects) showed a reduction of normal Y-bearing sperm, and an increase in nullisomic and XY-disomic sperm, suggesting an instability of the deleted Y chromosome causing its loss in germ cells, and meiotic alterations leading to XY non-disjunction. Intracytoplasmic injection of sperm from Y-deleted men will therefore transmit the deletion to male children, and therefore the spermatogenic impairment, but raises also concerns of generating 45,X and 47,XXY embryos.     

An improved flow cytometric assay for somatic mutations at the glycophorin A locus in humans

An improved method has been developed for the glycophorin A assay for somatic cell mutations in humans. The new assay, named the "BR6" assay, can be performed on a commercially available, single-beam flow cytometer, in contrast to the previously described 1W1 assay that required a dual-beam flow sorter. A modified cell labeling method developed for the BR6 assay results in improved separation of normal and mutant phenotype cells, as well as eliminating some cellular artifacts that affected the 1W1 assay. Parallel measurements on samples from 17 normal donors showed that the BR6 assay yields comparable variant cell frequencies and improved measurement precision compared with the 1W1 assay. A detailed analysis of three individuals who showed large differences in background variant frequency with the 1W1 assay confirmed that these differences could also be detected with the BR6 assay. A dramatically elevated variant cell frequency was seen with the BR6 assay of an individual exposed to a high level of ionizing radiation in an accident at Goiania, Brazil.     

Intervention of somatic mutational events in vivo by a germline defect at the adenine phosphoribosyltransferase locus

Both germline and somatic mutations are known to affect phenotypes of human cells in vivo. In previous studies, we cloned mutant peripheral blood T cells from germline heterozygous humans for adenine phosphoribosyltransferase (APRT) (EC 2.4.2.7) deficiency and found that approximately 1.3 × 10^–4 peripheral T cells had undergone in vivo somatic mutations. Loss of heterozygosity (LOH) was the major cause of the mutations at the APRT locus since approximately 80% of the mutant T cell clones exhibited loss of normal alleles. In the present study, we identified three heterozygous individuals for APRT deficiency (representing two separate families), in whom none of the somatic mutant cells exhibited LOH at the APRT locus. The germline mutant APRT alleles of these heterozygotes from two unrelated families had the same gross DNA abnormalities detectable by Southern blotting. None of the germline mutant APRT alleles so far reported had such gross DNA abnormalities. The data suggest that the germline mutation unique to these heterozygous individuals is associated with the abrogation of LOH in somatic cells. The absence of LOH at a different locus has already been reported in vitro in an established cell line but the present study describes the first such event in vivo in human individuals. Received: 10 May 1996     

Cooperative enhancement at the Drosophila Sgs-3 locus

The Drosophila glue gene Sgs-3 is specifically expressed in the secretory cells of the salivary glands of third instar larvae. We have assayed the expression of gene fusions to determine the role of cis-acting Sgs-3 sequences in conferring this pattern of expression. These experiments define two regulatory regions required for expression of reporter genes from the Sgs-3 promoter. One region, between 106 and 56 bp upstream of the Sgs-3 mRNA 5' end is sufficient for low but correct tissue- and stage-specific expression. A second region, lying between 629 and 130 bp 5' of the RNA start site is functionally equivalent; that is, it alone will also direct low level, specific expression. These two regions act synergistically to give high level expression. More distant upstream regions function to further increase levels of expression. These two regulatory elements can confer a salivary gland-specific pattern of expression on a heterologous promoter and are also sufficient to drive gene expression in other Drosophila species, implying conservation of regulators.     

TRA-1/GLI controls development of somatic gonadal precursors in C. elegans

TRA-1/GLI is best known as a master regulator of sex determination in the nematode C. elegans, but its fly and vertebrate homologs (e.g. Ci, GLI) regulate embryonic patterning and cell proliferation. In this paper, we show that TRA-1/GLI controls development of the two somatic gonadal precursors (SGPs) in both XX and XO animals, in addition to its role in sex determination. Normally, SGPs reside at the poles of the gonadal primordium and divide according to intrinsic gonadal axes. In tra-1-null mutants, however, SGPs assume non-polar positions and the polarity of one SGP is reversed. Consistent with its SGP function, TRA-1 protein is present in SGPs during embryogenesis and early larval development. Previous studies have shown that the ehn-3 gene also affects SGP positions, and we report here that tra-1 and ehn-3 interact genetically. Whereas SGPs in tra-1 and ehn-3 single mutants are largely normal and generate many descendants, those in tra-1; ehn-3 double mutants do not mature or divide. Furthermore, tra-1 is a dominant enhancer of the ehn-3 gonadal defect, which includes the enhancement of a weak sexual transformation in the gonad. We cloned ehn-3, and found that it encodes a C2H2 zinc-finger protein. A rescuing EHN-3::GFP reporter is predominantly nuclear and expressed specifically in SGPs. The EHN-3 protein is therefore likely to regulate gene expression. We propose that TRA-1/GLI and EHN-3 have overlapping roles in regulation of multiple steps of SGP development. We speculate that regulation of SGP development may be an evolutionarily ancient role of TRA-1/GLI in nematode development.     

Polarised complementation at the pyrimidine-3 locus of Neurospora

Summary A re-evaluation of complementation at the pyrimidine-3 locus ofNeurospora suggests the existence of polarity. This apparent polarity is supported by polar complementation of the majority of a number of mutants induced with the acridine ICR-170, and confirmed by the behavior of an allele which is shown by reversion studies to be a frameshift mutant. Supported by Public Health Service Grant AI-01462.