Molecular cloning of rat intestinal mucin. Lack of conservation between mammalian species

We have prepared antisera to deglycosylated rat intestinal mucin and used it to obtain immunoreactive clones from a rat jejunum cDNA library. Four of these clones were sequenced, and all were found to be partial cDNAs that contained 18-base pair tandem repeats characteristic of a mucin. These cDNAs encoded a repetitive peptide with a consensus sequence of TTTPDV. Thus, they bear little resemblance to either of the two human intestinal mucin cDNAs isolated previously (Gum, J. R., Byrd, J. C., Hicks, J. W., Toribara, N. W., Lamport, D. T. A., and Kim, Y. S. (1989) J. Biol. Chem. 264, 6480-6487 and Gum, J. R., Hicks, J. W., Swallow, D. M., Lagace, R. E., Byrd, J. C., Lamport, D. T. A., Siddiki, B., and Kim, Y. S. (1990) Biochem. Biophys. Res. Commun. 171, 407-415). One of these rat mucin clones, designated RMUC 176, was chosen for further analysis. This clone recognized a band of approximately 9 kilobases when used to probe RNA blots. A strong hybridization band was present using rat small intestine and colon RNA but was not detectable when RNA isolated from heart, liver, or kidney was tested. The RMUC 176 clone and the two previously isolated human intestinal mucin cDNA clones were used to probe blots prepared from BamHI-digested DNA of various species. Here, the human probes detected fragments present only in human and chimpanzee DNA, whereas the RMUC 176 clone recognized fragments only in rat and mouse DNA. Thus, the repetitive portions of intestinal mucin genes are apparently not well conserved between phylogenetically distant species.     

Molar growth yields and bioenergetic parameters of extremely alkaliphilic Bacillus species in batch cultures, and growth in a chemostat at pH 10.5.

Alkaliphilic Bacillus species that grow at pH 10.5 must cope with a low protonmotive force (-50 mV) due to a reversed transmembrane pH gradient at least 2 pH units more acid inside. Here we demonstrate that strictly alkaliphilic B. firmus RAB and two strains of B. alcalophilus (ATCC 27467 and DSM 485) grow exponentially in batch cultures with a doubling time of less than 1 h in 100 mM buffered medium, while the actual medium pH remains above 10.2. The ATCC strain continued to grow rapidly for at least 7 h, but the growth rate of the DSM strain declined dramatically after 3 h. However, both the B. alcalophilus strains, B. firmus RAB and facultatively alkaliphilic B. firmus OF4 were readily maintained for at least 24 h between pH 10.4 and 10.6 in a chemostat where nutrients were constantly replenished. A critical nutrient may be limiting in batch cultures of the DSM strain of B. alcalophilus. The facultative alkaliphile grew equally well in batch cultures at an initial pH of 7.5 or 10.5. Its molar growth yield (23 mg dry wt mmol-1) on malate (Ymal) was the same at the two pH values and was comparable to Ymal for B. subtilis grown at neutral pH. B. firmus RAB and B. alcalophilus ATCC 27467 grown at pH 10.5 also showed Ymal values at least as high as the neutralphile, indicating efficient use of the energy source even at low protonmotive force. Moreover, the phosphorylation potential of B. firmus OF4 grown at pH 7.5 (45.2 kJ mol-1) or pH 10.5 (46 kJ mol-1) was in a conventional range for bacteria.     

Dosage of the smallest chromosome affects both the yeast-hyphal transition and the white-opaque transition of Candida albicans WO-1.

The WO-1 strain of Candida albicans is capable of alternating between two highly distinct yeast cell types termed white and opaque (E. H. A. Rikkerrink, B. B. Magee, and P. T. Magee, J. Bacteriol. 170:895-899, 1988; B. Slutsky, M. Staebell, J. Anderson, L. Risen, M. Pfaller, and D. R. Soll, J. Bacteriol. 169:189-197, 1987). We have isolated WO-1 mutants that show a marked deficiency at being able to switch from the white form to the opaque form under conditions normally favorable for this transition. Pulsed-field electrophoresis demonstrated that one of the initial two spontaneous nonswitching mutants lacked the smallest chromosome that is normally present in WO-1. The availability of a WO-1 derivative whose only functional ADE2 gene is located on this small chromosome made possible, through the induction of chromosome nondisjunction, the isolation of numerous new mutants missing this chromosome as well as mutants containing two copies of the chromosome. Mutants missing the smallest chromosome showed a greatly diminished ability to produce opaque sectors and to produce germ tubes in the presence of human serum. Mutants containing two copies of the small chromosome showed an increased ability to produce germ tubes. These results indicate that this small chromosome carries one or more genes involved in both the white-opaque switch and the yeast-hyphal switch.     

Differential localizations of and requirements for the two Drosophila ninaC kinase/myosins in photoreceptor cells

The ninaC gene encodes two retinal specific proteins (p132 and p174) consisting of a protein kinase domain joined to a domain homologous to the head region of the myosin heavy chain. The putative myosin domain of p174 is linked at the COOH-terminus to a tail which has some similarities to myosin-I tails. In the current report, we demonstrate that the ninaC mutation results in light- and age-dependent retinal degeneration. We also show that ninaC flies display an electrophysiological phenotype before any discernible retinal degeneration indicating that the electrophysiological defect is the primary effect of the mutation. This suggests that ninaC has a role in phototransduction and that the retinal degeneration is a secondary effect resulting from the defect in phototransduction. To examine the requirements for the individual ninaC isoforms, mutant alleles were generated which express only p132 or p174. Elimination of p174 resulted in a ninaC phenotype as strong as the null allele; however, elimination of p132 had little if any effect. As a first step in investigating the basis for the difference in requirements for p174 and p132 we performed immuno-localization at the electron microscopic level and found that the two isoforms display different subcellular distributions in the photoreceptor cells. The p132 protein is restricted primarily to the cytoplasm and p174 to the rhabdomeres, the microvillar structure which is the site of action of many of the steps in phototransduction. This suggests that the p174 myosin-I type tail is the domain responsible for association with the rhabdomeres and that the substrate for the p174 putative kinase may be a rhabdomeric protein important in photo-transduction.     

Cleavage of Bovine Brain Microtubule-Associated Protein-2 by Human Immunodeficiency Virus Proteinase

The high-molecular-weight dendritic cytoskeletal protein known as microtubule-associated protein (MAP)-2 displays the capacity to stimulate tubulin polymerization and to associate with microtubules. Serine proteases cleave MAP-2 into a C-terminal M(r) 28,000-35,000 microtubule-binding fragment and a larger N-terminal M(r) 240,000 projection-arm region. We now show that human immunodeficiency virus (HIV) proteinase also progressively degrades purified MAP-2 in vitro. This proteolysis reaction is characterized by transient accumulation of at least six intermediates, and most abundant of these is an M(r) 72,000 species that retains the ability to associate with taxol-stabilized microtubules. Treatment of this M(r) 72,000 species with thrombin releases the same M(r) 28,000 component as that derived from thrombin action on intact high-molecular-weight MAP-2, indicating that the viral aspartoproteinase action preferentially occurs further toward the N-terminus. The association of the M(r) 72,000 component with microtubules can be disrupted by the presence of a 21-amino acid peptide analogue of the second repeated sequence in the MAP-2 microtubule-binding region. We also studied HIV proteinase action on MAP-2 in the presence of tubulin and other MAPs that recycle with tubulin, and contrary to other published studies we found no effect of such treatment on microtubule self-assembly behavior. Cleavage of isolated MAP-2 by the HIV enzyme at high salt concentrations, followed by desalting and addition of tubulin, also resulted in microtubule assembly, albeit with slightly reduced efficiency.     

The human MUC2 intestinal mucin has cysteine-rich subdomains located both upstream and downstream of its central repetitive region.

The human MUC2 mucin is a large secretory glycoconjugate that coats the epithelia of the intestines, airways, and other mucus membrane-containing organs. Previous work has shown that this mucin contains an extended tandem repeat-containing domain rich in Thr and Pro. In the present work we describe two additional regions of this mucin located both upstream and downstream of the tandem repeat array. The carboxyl-terminal domain contains 984 residues and can be divided into mucin-like (139 residues) and cysteine-rich (845 residues) subdomains. This latter subdomain exhibits varying degrees of sequence similarity to a wide range of mucins and mucin-like proteins including those isolated from rats, pigs, cows, and frogs. We also report here the sequence of 1270 residues lying immediately upstream of the tandem repeats. This region contains a repetitive, mucin-like subdomain and a second cysteine-rich stretch of more than 700 residues. Both cysteine-rich subdomains of this mucin have sequence similarity with von Willebrand factor, a serum protein that exists as a disulfide-linked polymer. This suggests that these cysteine-rich subdomains are important in the catenation of mucin monomers into oligomers, the structures that confer viscoelasticity upon mucus.     

Interactions between Pseudomonas aeruginosa and iodophor germicides in milking parlor udder wash water systems.

In a field study of 29 dairy farms, Pseudomonas aeruginosa was isolated more frequently (P = 0.05) from milking parlor udder wash water systems containing iodophor germicides than from those with no germicide. Most available iodine (AI2) concentrations were below the recommended level of 25 ppm (25 microgram/ml). Rubber and polyvinyl chloride hoses caused rapid decreases in the AI2 concentrations of 25 ppm iodophor solutions. AI2 dropped from 25 ppm to 6 ppm or less in 240 min for solutions contained in either polyvinyl chloride or rubber, compared with solutions in glass, which were unchanged in 240 min. Addition of inactivated iodophor solution to aqueous cultures resulted in significantly higher (P less than 0.05) numbers of P. aeruginosa at 10 and 24 h postinoculation. P. aeruginosa was grown in polyvinyl chloride tubing and exposed twice daily to 0, 10, or 25 ppm of AI2. None of the exposure concentrations eliminated the bacteria from the hoses, and bacterial numbers were not significantly different in hoses exposed to 0 and 10 ppm by the eighth treatment day. Bacteria taken from the water in these hoses were exposed to different concentrations of iodophor solution. Iodophor concentrations which will kill 50% of P. aeruginosa cultures previously exposed to 0, 10, and 25 ppm of AI2 were predicted to be 3.0, 11.8, and 20.8 ppm, respectively.     

Chromosomal rearrangements associated with morphological mutants provide a means for genetic variation of Candida albicans.

At frequencies as high as 1.4%, the pathogenic yeast Candida albicans spontaneously gave rise to morphological mutants exhibiting more than 20 different types of abnormal colonies; approximately two-thirds of the mutants were stable, while the other one-third were unstable and produced mixtures of different colonial forms at very high rates. Abnormal electrophoretic karyotypes were observed for all of the 14 mutants that were examined, indicating that they were associated with different types of single and multiple gross chromosomal rearrangements. Because C. albicans is asexual and does not go through a meiotic cycle, we suggest that the high frequency of chromosomal rearrangements provides a means for genetic variation in this organism.     

Molecular cloning of cDNAs derived from a novel human intestinal mucin gene

A human small intestinal lambda gt11 cDNA library was screened with antibodies to deglycosylated small intestinal mucin. Four partial cDNA clones were isolated that define a novel human mucin gene. These include two partial cDNA clones, SIB 124 and SIB 139, that contain 51 nucleotide tandem repeats which encode a seventeen amino acid repetitive peptide with a consensus sequence of HSTPSFTSSITTTETTS. SIB 139 hybridized to messages produced by small intestine, colon, colonic tumors and also by high mucin variant LS174T colon cancer cells. The gene from which cDNAs SIB 124 and SIB 139 are derived (proposed name MUC 3) maps to chromosome 7, distinct from other known human mucin genes.     

Species composition,similarity, and structure of mayan homegardens in tixpeual and tixcacaltuyub,yucatan, Mexico

We studied species composition, similarity, and structure of homegardens in two Yucatecan Maya communities, Tixpeual and Tixcacaltuyub, Yucatan, Mexico. The number of gardens sampled per village was 20 and 22; total area sampled was very similar, 45,265 m² and 40,150 m²; the number of trees and shrubs present was 5651 and 5603; and number of species was 135 and 133, respectively. Diversity was low for both sites (H′= 1.6), as were the correlation coefficients (r) for the species-area and individuals-area correlations. The relatively low values obtained for the structural parameters reflect the random pattern of plant incorporation to the gardens, the variability in the proportion of constantly used and not constantly used garden area, and a certain uniformity in the number of species used and number of individuals present, and the relationship between these parameters and garden size. All these reflect the uniqueness of each homegarden, which depends upon the cultural background of the owner. We noticed a trend towards a change in homegarden structure and function in response to the modernization process. Homegardens in villages in the outskirts of cities tend to have more ornamental species and commercial fruit plants than homegardens in isolated villages.