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.     

The malmö polymorphism of coagulation factor IX, an immunologic polymorphism due to dimorphism of residue 148 that is in linkage disequilibrium with two other F.IX polymorphisms

A mouse monoclonal antibody (MAB 9.9) to coagulation factor IX (F.IX) detects a polymorphism in the plasma of normal people. Its epitope has been narrowed down to <6 amino acids in the activation peptide of the X-linked F.IX protein. The activation peptide contains a dimorphism—Thr:Ala—at position 148 of the protein. Using synthetic oligonucleotides, we have demonstrated that (1) the F.IX which reacts with 9.9 has Thr at position 148 and (2) that which does not has Ala. Positive reactors (148^thr) are designated Malmö A, and negative reactors (148^ala) are designated Malmö B. The plasma levels of AA women are indistinguishable from those of A men, and both B men and BB women are null against MAB 9.9. The plasma level of Malmö A in AB women is approximately half that of AA women, and “lyonization” is clearly operating in the heterozygotes. The dimorphism is in strong linkage disequilibrium with two other intragenic RFLPs, TaqI and XmnI. Furthermore, intragenic crossing-over—including double crossing-over—appears to have occurred between the three sites. Seven of the eight possible haplotypes have been identified, five in men and two others in women. The immunoassay that identifies ～50% of the AB women in the pool of Malmö A females with 95% confidence identifies men unambiguously as A or B. The assay would be very useful for population-genetic studies of the Malmö epitope if the studies were limited to men.     

Examination of Genetic Relatedness of Marine Synechococcus spp. by Using Restriction Fragment Length Polymorphisms

The relatedness of several marine Synechococcus spp. was estimated by DNA hybridization. Strains isolated from various geographical locations and representing a diversity of DNA base compositions and phycobiliprotein profiles were compared by restriction fragment length polymorphisms for a number of genes. DNAs from two marine red algae and a cryptomonad alga (which exhibit a phycobiliprotein composition similar to that of the marine Synechococcus spp.) and Synechococcus strain PCC6301 (Anacystis nidulans) were also included in the comparison. Strains WH8008, WH8018, and WH7805 were shown to be very similar to one another, as were strains WH7802 and WH7803. Strains WH8110 and WH5701 were clearly unrelated to any of the other strains, and no marine Synechococcus isolate showed any similarity to the freshwater Synechococcus strain PCC6301 or the eucaryotic algae. The method is relatively straightforward and sensitive and uses a variety of basic molecular biology techniques. Its utility in ascertaining the genetic relatedness and diversity of marine Synechococcus spp. and possible extension to field studies are discussed.     

Rhizobium trifolii 0403 Is Capable of Growth in the Absence of Combined Nitrogen

Rhizobium trifolii 0403 was treated with 16.6 mM succinate and other nutrients and thereby induced to grow in nitrogen-free medium. The organism grew microaerophilically on either semisolid or liquid medium, fixing atmospheric nitrogen to meet metabolic needs. Nitrogen fixation was measured via ¹⁵N incorporation (18% ¹⁵N enrichment in 1.5 doublings) and acetylene reduction. Nitrogen-fixing cells had a K_m for acetylene of 0.07 atm (ca. 7.09 kPa), required about 3% oxygen for optimum growth in liquid medium, and showed a maximal specific activity of 5 nmol of acetylene reduced per min per mg of protein at 0.04 atm (ca. 4.05 kPa) of acetylene. The doubling time on N-free liquid medium ranged from 1 to 5 days, depending on oxygen tension, with an optimum temperature for growth of about 30°C. Nodulation of white clover by the cultures showing in vitro nitrogenase activity indicates that at least part of the population maintained identity with wild-type strain 0403.     

Target-sensitive immunoliposomes: preparation and characterization

A novel target-sensitive immunoliposome was prepared and characterized. In this design, target-specific binding of antibody-coated liposomes was sufficient to induce bilayer destabilization, resulting in a site-specific release of liposome contents. Unilamellar liposomes were prepared by using a small quantity of palmitoyl-immunoglobulin G (pIgG) to stabilize the bilayer phase of the unsaturated dioleoylphosphatidylethanolamine (PE) which by itself does not form stable liposomes. A mouse monoclonal IgG antibody to the glycoprotein D of Herpes simplex virus (HSV) and PE were used in this study. A minimal coupling stoichiometry of 2.2 palmitic acids per IgG was essential for the stabilization activity of pIgG. In addition, the minimal pIgG to PE molar ratio for stable liposomes was 2.5 X 10(-4). PE immunoliposomes bound with HSV-infected mouse L929 cells with an apparent Kd of 1.00 X 10(-8) M which was approximately the same as that of the native antibody. When 50 mM calcein was encapsulated in the PE immunoliposomes as an aqueous marker, binding of the liposomes to HSV-infected cells resulted in a cell concentration dependent lysis of the liposomes as detected by the release of the encapsulated calcein. Neither uninfected nor Sendai virus infected cells caused a significant amount of calcein release. Therefore, the release of calcein from PE immunoliposomes was target specific. Dioleoylphosphatidylcholine immunoliposomes were not lysed upon contact with infected cells under the same conditions, indicating that PE was essential for the target-specific liposome destabilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a two-signal-dependent, Ia+ mononuclear phagocyte progenitor subpopulation that is sensitive to inhibition by ferritin

Evidence is presented that the ferritin-inhibitable, Ia+ monocyte progenitor in murine marrow requires two signals for stimulation of clonal proliferation. Escherichia coli K235 lipopolysaccharide (LPS) at 0.1 ng/ml enhanced macrophage colony formation by 25 to 70% in murine marrow cultures stimulated with colony-stimulating factor (CSF-1). The progenitors which responded to LPS and CSF-1 represented a distinct subpopulation. Pretreatment of marrow cells with complement plus anti-Ia, anti-H2, anti-asialo GM1, and anti-Mac-1 antibodies specifically depleted the two-signal-requiring progenitors. In addition, the same progenitors were depleted by preincubation with hydroxyurea, indicating that these cells were in cell cycle when removed from the marrow. When compared with the quiescent progenitors, the Ia+, cycling cells were more sensitive to the antiproliferative effects of interferon alpha/beta but were more resistant to inhibition by E prostaglandins. Pretreatment with T cell-specific antibodies and complement specifically enhanced cloning of quiescent progenitors without affecting cloning of the Ia+, cycling subpopulation. Moreover, rat liver ferritin at 10(-8) to 10(-10) M specifically inhibited clonal proliferation of the Ia+ progenitors. Finally, the requirement for LPS as the additional stimulant could be replaced by the addition of haplotype-specific anti-Ia antibody to CSF-stimulated cultures. In contrast to LPS, anti-IA was competitive with inhibitory ferritin in clonal proliferation of the Ia+ progenitors. The significance of these observations in regulation of monocytopoiesis is discussed.     

Spontaneous cell shedding by tumor cells in monolayer culture

Summary Sarcoma 180 monolayers spontaneously shed single cells and small multicellular aggregates into the surrounding medium to produce a dual population of floating and substratum-attached cells. Shedding was a motility-associated event that occurred when cells attempted to migrate over one another. It resulted from a combination of cell shape change and active motility, which increased sensitivity to fluid shear dislodgement by reducing a cell's surface area of adhesive contact and increasing strain tension at its adhesive contact points. Shedding occurred at all phases of the cell cycle. Extracellular matrix but not conditioned medium enhanced the floating subpopulation by slowing the kinetics of rattachment to plastic and cellular substrata. Although sarcoma 180 cells are anchorage independent in the sense that they grow readily in single cell suspension, they nevertheless exhibited anchorage modulation of their cell cycle. Short periods in suspension produced a mild G₁ accumulation, whereas longer periods of anchorage deprivation led to a mild G₂ accumulation which appeared to result from an interference with cytokinesis. This work was supported by grants from the Medical Research Council of Canada, The National Cancer Institute of Canada, the Alberta Heritage Savings and Trust Fund for Applied Cancer Research, and the Alberta Heritage Fund for Medical Research.     

Effect of Metal-Rich Sewage Sludge Application on the Bacterial Communities of Grasslands

The effect of long-term application of heavy metal-laden sewage sludge on the total heterotrophic aerobic and the cadmium-resistant soil bacterial communities was studied. Gram-positive bacteria were completely absent from resistant communities. These findings suggest that this group is highly susceptible to Cd. Shannon's diversity indices estimated for total communities did not reveal negative effects on the communities that developed in the presence of sludge. However, Cd-resistant communities isolated from long-term sludge-amended soils were more diverse than the resistant communities from a control sample, suggesting that adaptation to Cd as a stressor had occurred in the presence of sludge constituents. This higher diversity was attributed to Cd resistance in pseudomonads and gram-negative fermenters. Resistance did not develop by dissemination of Cd resistance plasmids, because these were rarely detected in the genomes of resistant strains.