The human metallothionein gene cluster is not disrupted in myelomonocytic leukemia

The human metallothionein gene complex on chromosome 16 has been remapped to 16q13 using high-resolution in situ hybridization. The complex is not disrupted by the rearrangement breakpoint on the long arm of chromosome 16 in patients with myelomonocytic leukemia with abnormal eosinophils, as had been previously reported. The locus order on 16q is cen-MT-FRA16B-D16S4-inversion breakpoint-HP-tel.     

A new location for the human adenine phosphoribosyltransferase gene (APRT) distal to the haptoglobin (HP) and fra(16)(q23)(FRA16D) loci

The human adenine phosphoribosyltransferase gene (APRT) was mapped with respect to the haptoglobin gene (HP) and the fragile site at 16q23.2 (FRA16D). A subclone of APRT and a cDNA clone of HP were used for molecular hybridization to DNA from mouse-human hybrid cell lines containing specific chromosome 16 translocations. The APRT subclone was used for in situ hybridization to chromosomes expressing FRA16D. APRT was found to be distal to HP and FRA16D and was localized at 16q24, making the gene order cen-FRA16B-HP-FRA16D-APRT-qter.     

Genetic and physical mapping of a novel region close to the fragile X site on the human X chromosome

We report the isolation and characterization of a novel DNA marker (1A1) in Xqter in the region of the fragile X. Genetic studies in families segregating for the fragile X syndrome suggest that 1A1 lies between the disease mutation and the distal locus, DXS52. Studies in normal and fragile X families show that 1A1 is tightly linked to DXS52 (Zmax = 17.20; theta max = 0.03) and F8 (Zmax = 7.01; theta max = 0.08). Multipoint mapping of families supports the order Xcen-DXS105-FRAXA-1A1-DXS52-(F8, DXS115)-Xqter. Pulsed-field gel electrophoresis (PFGE) studies demonstrate that 1A1 defines a new region of at least 2 Mb of DNA not physically linked to DXS52 or F8, thus extending the physical map of Xq27-qter to over 4 Mb. Complex partial digestion PFGE patterns, probably due to differing degrees of methylation, are observed with 1A1 in unrelated normal and fragile-X-positive individuals, whereas other distal markers give uniform digestion profiles. Physical data suggest that 1A1 lies in a region less CpG rich than other distal markers in Xq27-qter.     

Chromosomal localization of ARSB,the gene for human N-acetylgalactosamine-4-sulphatase

Summary A deficiency of N-acetylgalactosamine-4-sulphatase (G4S, gene symbol ARSB), results in the accumulation of undegraded substrate and the lysosomal storage disorder, Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI). In situ hybridization using an ³H-labelled human G4S genomic DNA fragment to human metaphase chromosomes localized ARSB to chromosome 5q13–5q14. This location is consistent with, an refines, previous chromosomal assignments based on the expression of human G4S in somatic cell hybrids.     

Effects of the epoxide hydrase inhibitor, 1,1,1-trichloropropane-2,3-oxide on the genetic activity of aflatoxin B1 metabolites in in vitro activation test systems.

The epoxide hydrase inhibitor 1,1,1-trichloroprophane-2,3-oxide (TCPO) was genetically active to cells of S. cerevisiae and conidia of N. crassa. This genetic activity could be eliminated or reduced to near spontaneous levels in the presence of the S-9 fraction of hamster liver homogenate. The addition of TCPO to an in vitro activation system containing aflatoxin B1 resulted in an increase in the genetic activity of aflatoxin B1, and this increase was dependent on the dose of TCPO. These results are discussed in relation to the possible metabolism of the promutagen aflatoxin B1.     

Physical mapping of new DNA probes near the fragile X mutation (FRAXA) by using a panel of cell lines

The fragile X syndrome is a very common disorder, but there has been little progress toward isolating the fragile X mutation (FRAXA). We describe a panel of 14 somatic cell hybrid lines, lymphoblastoid cell lines, and peripheral lymphocytes with X-chromosome translocation or deletion breakpoints near FRAXA. The locations of the breakpoints were defined with 16 established probes between pX45d (DXS100) and St14-1 (DXS52). Seven of the cell lines had breakpoints between the probes RN1 (DXS369) and U6.2 (DXS304), which flank FRAXA at distances of 3-5 centimorgans. The panel of cell lines was used to localize 16 new DNA probes in this region. Six of the probes-VK16, VK18, VK23, VK24, VK37, and VK47--detected loci near FRAXA, and it was possible to order both the X-chromosome breakpoints and the probes in relation to FRAXA. The order of probes and loci near FRAXA is cen-RN1,VK24-VK47-VK23-VK16,FRAXA-++ +VK21A-VK18-IDS-VK37-U6.2-qter. The breakpoints near FRAXA are sufficiently close together that probes localized with this panel can be linked on a large-scale restriction map by pulsed-field gel electrophoresis. This panel of cell lines will be valuable in rapidly localizing other probes near FRAXA.     

An expanded mouse-human hybrid cell panel for mapping human chromosome 16

A mouse/human hybrid cell panel of human chromosome 16 has been extended to a total of 31 hybrids. These hybrids were derived from constitutional translocations and deletions ascertained during clinical cytogenetic studies. This panel of hybrids, together with four fragile sites, have the potential to divide chromosome 16 into 38 regions. Rapid detailed physical mapping of gene probes or anonymous DNA probes is possible using this hybrid panel. This hybrid cell panel also allows the physical mapping of other chromosomes with three breakpoints on chromosomes 1, 4, 11 and 13 and two on chromosomes 3, 10 and 18.     

A Highlights from MBoC Selection: Protein kinase C activation decreases peripheral actin network density and increases central nonmuscle myosin II contractility in neuronal growth cones

Protein kinase C (PKC) can dramatically alter cell structure and motility via effects on actin filament networks. In neurons, PKC activation has been implicated in repulsive guidance responses and inhibition of axon regeneration; however, the cytoskeletal mechanisms underlying these effects are not well understood. Here we investigate the acute effects of PKC activation on actin network structure and dynamics in large Aplysia neuronal growth cones. We provide evidence of a novel two-tiered mechanism of PKC action: 1) PKC activity enhances myosin II regulatory light chain phosphorylation and C-kinase–potentiated protein phosphatase inhibitor phosphorylation. These effects are correlated with increased contractility in the central cytoplasmic domain. 2) PKC activation results in significant reduction of P-domain actin network density accompanied by Arp2/3 complex delocalization from the leading edge and increased rates of retrograde actin network flow. Our results show that PKC activation strongly affects both actin polymerization and myosin II contractility. This synergistic mode of action is relevant to understanding the pleiotropic reported effects of PKC on neuronal growth and regeneration.     

Galactomannanases Man2 and Man5 from Thermotoga species: growth physiology on galactomannans, gene sequence analysis, and biochemical properties of recombinant enzymes

The enzymatic hydrolysis of mannan-based hemicelluloses is technologically important for applications ranging from pulp and paper processing to food processing to gas and oil well stimulation. In many cases, thermostability and activity at elevated temperatures can be advantageous. To this end, the genes encoding beta-mannosidase (man2) and beta-mannanase (man5) from the hyperthermophilic bacteria Thermotoga neapolitana 5068 and Thermotoga maritima were isolated, cloned, and expressed in Escherichia coli. The amino acid sequences for the mannosidases from these organisms were 77% identical and corresponded to proteins with an M(r) of approximately 92 kDa. The translated nucleotide sequences for the beta-mannanase genes (man5) encoded polypeptides with an M(r) of 76 kDa that exhibited 84% amino acid sequence identity. The recombinant versions of Man2 and Man5 had similar respective biochemical and biophysical properties, which were also comparable to those determined for the native versions of these enzymes in T. neapolitana. The optimal temperature and pH for the recombinant Man2 and Man5 from both organisms were approximately 90 degrees C and 7.0, respectively. The presence of Man2 and Man5 in these two Thermotoga species indicates that galactomannan is a potential growth substrate. This was supported by the fact that beta-mannanase and beta-mannosidase activities were significantly stimulated when T. neapolitana was grown on guar or carob galactomannan. Maximum cell densities increased by at least tenfold when either guar or carob galactomannan was added to the growth medium. For T. neapolitana grown on guar at 83 degrees C, Man5 was secreted into the culture media, whereas Man2 was intracellular. These localizations were consistent with the presence and lack of signal peptides for Man5 and Man2, respectively. The identification of the galactomannan-degrading enzymes in these Thermotoga species adds to the list of biotechnologically important hemicellulases produced by members of this hyperthermophilic genera.