Isolation and regional localization of a large collection (2,000) of single-copy DNA fragments on human chromosome 3 for mapping and cloning tumor suppressor genes

Summary A collection of 2,000 lambda phage-carrying human single-copy inserts (> 700 bp) were isolated from two chromosome-3 flow-sorted libraries. The single-copy DNA fragments were first sorted into 3p and 3q locations and about 700 3p fragments were regionally mapped using a deletion mapping panel comprised of two humanhamster and two-human-mouse cell hybrids, each containing a chromosome 3 with different deletions in the short arm. The hybrids were extensively mapped with a set of standard 3p markers physically localized or ordered by linkage. The deletion mapping panel divided the short arm into five distinct subregions (A-E). The 3p fragments were distributed on 3p regions as follows: region A, 26%; B, 31%; C, 4%; D, 4% and E, 35%. We screened 300 single-copy DNA fragments from the distal part of 3p (regions A and B) with ten restriction endonucleases for their ability to detect restriction fragment length polymorphisms (RFLPs). Of these fragments 110 (36%) were found to detect useful RFLPs: 35% detected polymorphisms with frequency of heterozygosity of 40% or higher, and 25% with frequency of 30% or higher. All polymorphisms originated from single loci and most of them were of the base pair substitution type. These RFLP markers make it possible to construct a fine linkage map that will span the distal part of chromosome 3p and encompasses the von Hippel-Lindau disease locus. The large number of single-copy fragments (2,000) spaced every 100–150 kb on chromosome 3 will make a significant contribution to mapping and sequencing the entire chromosome 3. The 300 conserved chromosome 3 probes will increase the existing knowledge of man-mouse homologies.     

Molecular characterization of a patient with del(1)(q23–q25)

Summary We report a patient (S.T.) with multiple congenital anomalies and developmental delay associated with an interstitial deletion of 1q23–1q25. Molecular analysis of the deletion was performed using DNA markers that map to 1q. Five DNA markers, MLAJ-1 (D1S61), CRI-L1054 (D1S42), HBI40 (D1S66), OS-6 (D1S75), and BH516 (D1S110), were demonstrated to be deleted. Informative polymorphisms demonstrated this to be a de novo deletion of the maternally derived chromosome. Deletion status was determined using restriction fragment length polymorphism (RFLP) analysis supplemented with densitometry in the experiments where RFLP analysis was not fully informative. Deletions were confirmed by Southern analysis using genomic DNA from a somatic cell hybrid retaining the del(1)(q23–q25) chromosome that was constructed from patient S.T. Flow karyotyping confirmed the deletion and estimated that the deletion encompassed 11,000–16,000 kb. The clinical and cytogenetic characteristics of S.T. are compared with those of ten previously described patients with monosomy 1q21–1q25.     

Mapping of mouse gamma crystallin genes on chromosome 1

Restriction fragments analysis of DNA from mouse-hamster somatic-cell hybrid clones revealed that a mouse gamma crystallin cDNA hybridized to genomic sequences located on mouse chromosome 1. Identification of restriction fragment length polymorphisms (RFLPs) in the gamma crystallin sequences of inbred strains of mice permitted the further localization of the gamma crystallin genes (Cryg) to the proximal region of chromosome 1 closely linked to the loci encoding isocitrate dehydrogenase (Idh-1), a low molecular weight (LM) crystallin protein polymorphism (Len-1), and fibronectin (Fn-1). A single recombinant was observed betweenLen-1 and an RFLP in the gamma crystallin gene family, consistent with the hypothesis thatLen-1 is one of the several structural loci encoding gamma crystallin genes.Len-1 is probably located on the centromeric end of theCryg gene family. Linkage ofIdh-1, Cryg, andFn-1 in mice extends the syntenic relationship of those loci to the human, bovine, and rodent genomes and may define a chromosomal region that is generally conserved among mammals. The map position ofCryg, near the eye lens obsolescence (Elo) locus, was confirmed by the discovery that the restriction fragment patterns of gamma crystallin sequences differed between strain C3H/HeJ and the congenic anophthalmic mutant strain, C3H.Elo. Therefore, the gamma crystallin genes were contransferred with the mutantElo gene in the derivation of C3H.Elo. The results establish that LEN-1 is a marker for the gamma crystallin gene family, position the gamma crystallin gene family relative to other markers on mouse chromosome 1, and provide additional evidence that theElo mutation is encoded at a locus closely linked to the gamma crystallin gene cluster. This study found no evidence of recombination hot spots within the gamma crystallin gene cluster.     

Influence of DNA sequence on the nature of mispairing during DNA synthesis

A series of synthetic oligonucleotide primers, annealed at various positions along the lacZ-alpha region of bacteriophage M13mp9 template, were elongated by purified DNA polymerases in the presence of only 3 of the 4 deoxynucleoside triphosphates to achieve misincorporation at a total of 49 different positions along the template. The newly synthesized strands (containing misincorporated bases) were isolated and sequenced to determine the identity of misincorporated deoxynucleoside monophosphates. The results indicate that the kind of mispairing that occurs during DNA synthesis is greatly influenced by the nucleotide sequence of the template. Transition-type base substitutions predominated overall, but at many template positions, transversion-type base substitutions occurred, most commonly via A.A mispairing. The results of parallel determinations made with Escherichia coli DNA polymerase I ("large fragment" form) and DNA polymerase of Maloney murine leukemia virus indicated that, overall, the identity of polymerase had only a small effect on the kind of misincorporation that occurred at different positions along the template. However, at certain template positions, the nature of mispairing during DNA synthesis was reproducibly affected by differing polymerase active-site environment.     

Aggressive chemotherapy in adult primary myelodysplastic syndromes. A report on 29 cases

Twenty-nine adult patients with primary myelodysplastic syndromes (MDS) and an excess of marrow blasts were treated by aggressive chemotherapy while still in MDS phase (20 cases) or after progression to ANLL (9 cases). Median age was 47.5 (range 18-68). Twenty-eight patients received a combination of Rubidazone and Ara C and 1 received High dose Ara C. Fourteen patients (48%) achieved complete remission (CR), 5 (17%) were treatment failures (F) and 10 (35%) died during therapy induced aplasia (DA). Median disease free survival was 8.5 months. Median survival of the whole population was 6 months from the onset of treatment, and 17 months in patients achieving CR. These results were significantly less favorable than those obtained at our institution in de novo ANLL with the same chemotherapy regimens. No statistically significant prognostic factors of treatment outcome emerged but patients with normal cytogenetic findings seemed to have both a higher CR rate and longer remissions than patients with abnormal karyotypes. Patients under 50 did not have higher CR rates than older patients, although they had longer remissions (with 3 out of 6 CRs exceeding 2 years). Finally, treatment outcome and survival were identical in patients treated in the MDS phase and in those treated after progression to ANLL. Combination chemotherapy is a highly toxic approach in MDS and essentially seems to benefit younger patients with a normal karyotype, in whom some long remissions can be obtained.     

Peripheral and integral subunits of the tonoplast H+-ATPase from oat roots

The subunit organization of the tonoplast H+-pumping ATPase from oat roots (Avena sativa L. var. Lang) was investigated. Tonoplast vesicles were treated with low ionic strength solutions (0.1 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer or 0.1 mM Na EDTA), carbonate, or a chaotropic reagent (KI), and then centrifuged to give a soluble fraction and a pellet. Treatments with low ionic strength solutions or KI resulted in 70-80% reduction in the membrane-associated ATPase activity, but did not affect the K+-stimulated pyrophosphatase activity. Polypeptides of 72, 60, and 41 kDa were solubilized from tonoplast vesicles by these wash treatments. These polypeptides reacted with polyclonal antibodies against the holoenzyme of tonoplast ATPase (anti-ATPase) and copurified with the tonoplast ATPase activity during gel filtration chromatography (Sepharose CL-6B). Mono-specific antibody against the 72- or 60-kDa polypeptide reacted with the solubilized 72- or 60-kDa polypeptide, respectively. However, the N,N-[14C]dicyclohexylcarbodiimide-binding 16-kDa polypeptide and a 13-kDa polypeptide that also reacted with anti-ATPase and copurified with the tonoplast ATPase activity during gel filtration remained in the pellets after the wash treatments. We conclude that the 72- and 60-kDa polypeptides appear to be peripheral subunits of the tonoplast ATPase and that the 16-kDa polypeptide is probably embedded in the membrane bilayer. Additional subunits of the ATPase complex may include a 41-kDa (peripheral) and a 13-kDa (integral) polypeptide. Based on these results, a working model of the tonoplast ATPase analogous to the F1F0-ATPase is proposed.     

Fetal-maternal transfer of [H3]-6 beta-hydroxycortisol in the pregnant ewe

Distribution and fetomaternal transfer of 6 beta-hydroxycortisol (6 beta-OHF) was studied using serial sampling following injection of tritium labelled 6 beta-OHF into various fluid compartments in the chronically cannulated unaesthesized pregnant ewe. There was a rapid transfer of 6 beta-OHF from the fetal circulation into amniotic fluid and maternal blood. In contrast, the maternal----fetal transfer of this steroid metabolite was considerably less. The sequence of appearance of 6 beta-OHF in fetal blood and amniotic fluid following injection into maternal blood suggests that this steroid is first transferred across the placenta to fetal blood before gaining entry into the amniotic fluid space. The half-lives of 6 beta-OHF after initial equilibration in maternal plasma, fetal plasma and amniotic fluid were 2.0 h, 5.1 h and 8.9 h respectively. The amniotic sac appears to contain a relatively static pool of 6 beta-OHF and may act as a "trap" for 6 beta-OHF in the ovine conceptus.     

Phosphorylation of the mouse hepatitis virus nucleocapsid protein

Analysis of the radiolabeled tryptic peptides derived from the nucleocapsid proteins of two serotypes of mouse hepatitis virus showed each to have a small number of unique peptides; however, two biologically distinct variants of the JHM strain appeared identical. Analysis of [32P]-labeled nucleocapsid-derived peptides showed that phosphorylation occurs at only a few sites and that all three viruses differed in the sites of phosphorylation. No differences in the sites of phosphorylation were found between the nucleocapsid proteins derived from purified virions and the membranes or the cytosol of infected cells, suggesting that post-translational phosphorylation plays no role in the regulation of viral assembly. These data show unequivocal evidence that the nucleocapsid proteins of mouse hepatitis virus strains differ in the sites of phosphorylation.     

Immunohistochemical localization of two angiotensin I-converting isoenzymes in the reproductive tract of the male rabbit

The male reproductive tract contains two different isoenzymes of angiotensin I-converting enzyme (ACE), i.e., pulmonary and testicular ACE. The present study shows selectively the cellular distribution of the ACE isoenzymes in the reproductive tract of male rabbit, using indirect immunofluorescence or immunoperoxidase methods. Testicular ACE was found in the seminiferous tubules of the testes in spermatocytes containing mature spermatids, and in spermatids within the epididymal tubular lumen in sexually mature, but not in immature, rabbits. Epididymal tubular cells contained pulmonary ACE. In the young rabbit, epididymal tissue contained more ACE than that in adult rabbit, since ACE was observed in principal cells in addition to basal cells. In mature rabbit, ACE was observed in basal cells only. Strong staining for pulmonary ACE was observed in cells of the vas deferens in both young and adult rabbit. Therefore, synthesis of epididymal ACE, unlike the testicular isoenzyme, was not stimulated by sexual maturation. Enzymatically active ACE in seminal fluid corresponds to the pulmonary isoenzyme. The present study indicates that this seminal fluid ACE may originate from cells of the epididymal tubules, particularly those of the vas deferens. Endothelial cells of blood vessels lying in the interstitium of both testicular and epididymal tissue contained the pulmonary isoenzyme.     

Seed dispersal and seedling emergence in an old field community in central New York (USA)

Summary Seed dispersal and seedling emergence of common taxa growing in a Solidago-dominated old field in central New York (USA) were monitored from May 1982 to June 1984. Over 3.5x10⁴ seeds per m² were captured on seed traps in each of the two years, with peaks occuring in July (due to Hieracium) and in November (due to Solidago). About 4.0x10³ seedling/m² emerged beneath the intact community in each of the two years. Although seedlings emerged predominantly in the early spring, a secondary peak occurred in September and October when many seedlings of introduced grasses appeared. Two additional aspects of the reproductive biology of the major taxa were related to the seasonal timing of seed dispersal: As the date of peak seed dispersal (among taxa) became progressively later in the season, (1) the duration of dispersal increased from about one week to about one-half year, and (2) the delay between the peak of seed dispersal and the peak of seedling emergence increased from a few days to about one-half year.