Differentiation of HT-29 human colonic adenocarcinoma cells correlates with increased expression of mitochondrial RNA: effects of trehalose on cell growth and maturation.

The HT-29 human adenocarcinoma cell line has been used extensively in the study of colonic cell differentiation and colon cancer. We report here that substitution of glucose with trehalose (alpha-D-glucopyranosyl-alpha-D-glucopyranoside) depresses growth and promotes mucin-producing, goblet-like maturation of HT-29. An initial characterization of this process was made by analyzing several cDNA clones whose RNA templates were differentially expressed at elevated levels in cells grown in trehalose-containing medium. Seven of the 9 clones examined corresponded to 6 mitochondrial genes whose expression levels, relative to those from glucose-grown cells, ranged from approximately 3-fold for 16S rRNA to 8-23-fold for NADH dehydrogenase subunit 4. On the other hand, levels of mitochondrial DNA copy, measured by using NADH dehydrogenase subunit 4 cDNA as probe, were shown to be unaffected by trehalose treatment. Elevation of cellular NADH dehydrogenase subunit 4 RNA in HT-29 cultures grown in medium containing different components (sodium butyrate, galactose, no-sugar, glucose, cellobiose) generally correlated with depressed growth levels and specifically with increased numbers of mucin-producing cells present. Like butyrate, the sugar, trehalose, is an effective inducer of HT-29 differentiation, and may prove useful as a dietary therapeutic, and as a probe for elucidating mitochondrial involvement in colonic cell differentiation and transformation.

Expression of Xenopus laevis histone H5 gene in yeast.

As an approach to assess linker histone function, we engineered a cDNA encoding Xenopus laevis histone H5 (XLH5), into the yeast Saccharomyces cerevisiae, which lacks any known proteins homologous to linker histones. XLH5 cDNA when fused to the yeast GAL10 promoter and 5' untranslated region (UTR) was shown to be accurately transcribed at relatively high levels in cells harvested at mid to late log after exposure to at least 22 mM galactose. The resultant 0.95 kb XLH5 transcript reached steady state levels by approx. 2 h after galactose induction. In contrast, the product, detected by anti-XLH5 antibody, was not stably expressed until 4 h or more after induction, when no apparent growth takes place. The expression product was 27% smaller than native H5 and may have been proteolytically processed. Constitutive transcription and loss of XLH5 expression product occurred using a plasmid construct containing a 275 bp fragment of the pBR322 tetr gene inserted downstream of the GAL10 promoter. This fragment carries a putative yeast cell-type-specific upstream activation sequence.

A temporally expressed gene from Schwanniomyces alluvius and detection of homologous sequences in other yeasts.

The boundaries of a temporally expressed gene (EG1) located on a 6.5-kb HindIII chromosomal DNA fragment of the yeast Schwanniomyces alluvius have been defined by cDNA hybridization and S1 nuclease mapping. The sequence of this gene reveals a 474-bp open reading frame (158 aa) and a TATA-like sequence 100 bp upstream from the first start codon. The encoded protein bears little resemblance with other known yeast structural proteins, including histones. A portion of the 3' untranslated region shows 50% identity with a consensus repetitive sequence (B2) found in the mouse genome. Repetitive sequences are also located in both 5' and 3' untranslated regions. Genomic DNA analysis indicated that related sequences were detected at two loci in Schw. alluvius and one locus in Saccharomyces cerevisiae. In contrast to the continuous transcription pattern of the ADE1 genes of Schw. alluvius and S. cerevisiae, the 0.8-kb transcripts of EG1 in these yeasts were most abundant in cells in early-log phase of growth and absent in cells of late-log phase, suggesting discontinuous synthesis.

Mitochondrial RNA abundance in differentiating human colonic epithelial tumor cells estimated through use of a mitochondrial genome map.

We describe a procedure for quantification of mitochondrial (mt) RNA present in total RNA extracts of HT-29 human colonic adenocarcinoma cells grown under conditions for rapid growth (25 mM glucose) or differentiation (25 mM trehalose or 5 mM butyrate). Purified mt DNA was fragmented into specific coding regions using restriction endonuclease sites predicted from HeLa cell mt DNA sequence and probed with either 32P-labelled mt DNA or cDNA made from total RNA of HT-29 or HeLa cells. The amounts of probe that hybridized to various gene-encoded mt DNA fragments or RNA were quantified by laser densitometry. Use of 13 restriction endonucleases revealed that most if not all the mt DNA of HT-29 and K562 leukemic cells was comparable in size to that of HeLa cells. Relative levels of mt RNA from rapidly growing HT-29 and HeLa cells were lower than those measured for differentiated HT-29 cells induced by either trehalose or butyrate. In rapidly growing HT-29 cells and HeLa cells, the highest levels of specific mt RNAs were those encoded by mt DNA sequences immediately flanking the nested promoters and the heavy-strand replication origin (OH). Expression patterns of specific mt RNAs from HT-29 cells treated with butyrate and with trehalose were similar, but not identical. In either case, the mt RNAs that increased the most were those coded by mt DNA sequences located downstream from the light-strand replication origin (OL), suggesting a novel pattern of expression not seen before.

Molecular cloning and characterization of a late Tipula iridescent virus gene.

Virions of the cytoplasmic, icosahedral insect virus, Tipula iridescent virus (TIV), contain two major DNA components (L, greater than 176 kb; and S1, 10.8 kb) and 25-30 proteins. We characterized a gene (L96) whose 3.6-kb transcript is expressed late in the course of TIV infection of cultured of Estigmene acrea (salt marsh caterpillar, permissive host) and Aedes albopictus (mosquito, semipermissive host) cells. The L96 gene has an open reading frame of 867 codons, predicting a protein of 96 kDa with a pI of 10.9. The C terminus of the L96 protein is rich in hydrophobic amino acids and contains a small region of homology spanning a proteolytic cleavage site within two mammalian viral (GAG) polyproteins. Additional identity with H5 lysine-rich histones in the same region and with other DNA-binding proteins suggests that this protein may be involved in TIV structure. The lengths of the 5'- and 3'-untranslated regions of the L96 transcript were determined to be 21 nucleotides (nt) and 700 nt, respectively. Comparison of the TIV L96- and capsid-encoding genes, both of which are expressed late in infection, revealed that their 5' and 3' regions are generally rich in A and T residues, and that their 3' ends encode at least one eukaryotic polyadenylation signal (AATAAA).

Molecular cloning of the ADE1 gene of Saccharomyces cerevisiae and stability of the transformants.

Plasmid YEp ( ADE1 )1a, containing a 2.7-kb Sau3A fragment of Saccharomyces cerevisiae DNA inserted at the BamHI site of the yeast shuttle vector pBTI -1 (Morris et al., 1981), results in high frequency, unstable transformation of ade 1 yeast strains. A second plasmid, YRp ( ADE1 )2, containing adjacent 0.5-kb and 3.0-kb BamHI fragments in pBR322 gave three types of yeast transformants: (1) transformants carrying extrachromosomal copies of the plasmid which indicate the presence of a functional ars sequence, (2) transformants indistinguishable from ade 1 strains by hybridization analysis, and (3) a transformant carrying a multimeric form of YRp ( ADE1 )2. Cells transformed with either of the plasmids are free of the red pigment characteristic of ade 1 mutants and indicate potential for direct colour-based selection of yeast transformants using ADE1 plasmids.

Aspergillus oryzae has two nearly identical Taka-amylase genes, each containing eight introns.

cDNA and genomic DNA for two nearly identical genes, AmyI and AmyII, coding for the enzyme Taka-amylase A (TA-A) of the fungus Aspergillus oryzae have been cloned and characterized. These genes are apparently unlinked, differing by only 3 nucleotides (nt) out of the 2720 nt that span the coding regions. The 617-nt 5'-flanking regions differ only at nt -372 (T or A) from the putative ATG start codon and contain four sets of short, inverted repeats (IR) upstream from the putative TATAAA box at nt -100 and the transcription start point at nt -69. The coding regions consist of 499 codons disrupted by eight intervening sequences. The putative proenzymes differ by only two amino acids (aa) and consist of the 478-aa extracellular enzyme plus a 21-aa hydrophobic leader sequence. Except for the replacement site changes in codons 35 (Arg----Gln) and 151 (Phe----Leu), the identity of the two genes continues downstream for 58 nt past the TGA stop codon before diverging. Exon 9 codes for 94 of the 98 aa of the domain B of mature TA-A. Little conservation of TA-A exons was found when these exons were aligned with those of human amylase. The genes are flanked by at least 6 to 10 kb of unrelated chromosomal nucleotide sequence. The Amy genes are co-expressed, since mRNA (cDNA) specific to the 3'-UTR of both genes was recovered from mycelia grown on starch, a known inducer of TA-A biosynthesis. The 3'-UTRs of cDNAs related to AmyI are shorter (128 nt and 145 nt) than those of AmyII (179 nt and 297 nt). The AmyI specific 3'-UTR is characterized by the absence of IR sequences and the presence of a putative 'AATAAA' polyadenylation signal.

Isolation and expression of cDNA clones coding for two sequence variants of Xenopus laevis histone H5.

We have cloned and characterized cDNAs coding for two variants of Xenopus laevis H5 histone protein (previously called H1s). cDNA was synthesized from RNA of immature erythrocytes in a single reaction using a modification of the method of Gubler and Hoffman [Gene 25 (1983) 263-269], and blunt-end ligated into the HincII site of the phage vector M13mp9. Immunological screening with a polyclonal antibody yielded two clones expressing H5 peptide. Sequence characterization revealed that both clones contained partial cDNA inserts and that the smaller 340-bp clone initiated reverse transcription within the coding region, at a site rich in adenine. Rescreening of the cDNA bank by nucleic acid hybridization produced eleven additional H5 clones, one of which coded for a second variant of H5. These two variants, called XLH5A and XLH5B, are very similar in sequence and code for proteins of 195 and 193 amino acids, respectively, which may be the H1D and H1E variants observed previously. XLH5, avian H5 and human H1O share identity at both nucleotide and amino-acid sequence levels. Further, the XLH5-coding mRNA is likely polyadenylated and lacks the highly conserved, 23-nucleotide dyad symmetry element found within the 3' untranslated regions of most histone-coding mRNAs.

Complete nucleotide sequence and mRNA-mapping of the large subunit gene of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Chlamydomonas moewusii.

Nucleotide (nt) sequence of the large subunit (LS) gene of ribulose-1,5-bisphosphate carboxylase/oxygenase from the green alga, Chlamydomonas moewusii, and mapping of transcription ends was achieved by two new strategies. The deduced LS sequence of 475 amino acid residues was compared with similar genes from six other species; cyanobacteria, land plants and a related alga (C. reinhardtii). The most conserved regions are the three ribulose bisphosphate binding sites and the CO2 activator site. The nt sequence conservation outside the coding region is limited to only three segments within the 5'-flanking region: a region of tandem repeats, TATAA box and ribosome-binding site. Termination point of transcription is an 'A' residue 3' to the first of two 18-nt inverted repeats, which has the potential to form a stem-loop hairpin structure. The possible role of these potential regulatory features for transcription and translation, and similar structures in other LS genes is presented.

Rapid synthesis and cloning of complementary DNA from any RNA molecule into plasmid and phage M13 vectors.

We describe several modifications of the Gubler and Hoffman procedure [Gene 25 (1983) 263-269] for complementary DNA (cDNA) synthesis that expand the versatility of this method for the rapid synthesis and cloning of double-stranded (ds) cDNA. These modifications include: (1) The combination of first and second strand synthesis into a single two-step reaction, which reduces the time for synthesis of blunt-ended ds-cDNA to less than 4 h. (2) The use of random hexadeoxyribonucleotide primers (RP) for the synthesis of ds-cDNA, which allows the synthesis of cDNA from any RNA template. (3) The combined use of random primers and DNA ligase treatment of cDNA/RNA hybrids prior to second-strand synthesis, which promotes the production of nearly full length ds-cDNA molecules. (4) The use of gel filtration to size-fractionate ds-cDNA, which allows the selection of specific size classes of ds-cDNA for cloning. (5) The use of blunt-end ligation to insert the ds-cDNA into the vector, which reduces the total time required for the construction of cDNA libraries to less than 24 h.

Applications of recombinant DNA technology to the pulp and paper industry.

New gene selection techniques (Recombinant DNA) are currently available to exploit useful properties of various biological systems hitherto regarded as interesting but of little or no immediate commercial value. The application of genetic engineering techniques to problems in the Pulp and Paper Industry are many. As a first step these techniques are being used to provide much needed fundamental information on the cellular and molecular mechanisms involved in the expression of extra-cellular enzymes that degrade lignocellulosic pulping wastes. The information gleaned from the studies on cellulolytic fungi and bacteria can be used to genetically engineer a yeast or bacterium capable of converting pulping wastes into ethanol and other useful by-products.

Human cell exposure assays of Bacillus thuringiensis commercial insecticides: production of Bacillus cereus-like cytolytic effects from outgrowth of spores.

Most contemporary bioinsecticides are derived from scaled-up cultures of Bacillus thuringiensis subspecies israelensis (Bti) and kurstaki (Btk), whose particulate fractions contain mostly B. thuringiensis spores (> 10(12)/L) and proteinaceous aggregates, including crystal-like parasporal inclusion bodies (PIB). Based on concerns over relatedness to B. cereus-group pathogens, we conducted extensive testing of B. thuringiensis (BT) products and their subfractions using seven human cell types. The Bti/Btk products generated nonspecific cytotoxicities involving loss in bioreduction, cell rounding, blebbing and detachment, degradation of immunodetectable proteins, and cytolysis. Their threshold dose (Dt approximately equal.5 times 10(-14)% BT product/target cell) equated to a single spore and a target cell half-life (tLD(50)) of approximately 16 hr. At Dts > 10(4), the tLD(50) rapidly shifted to < 4 hr; with antibiotic present, no component, including PIB-related [delta]-endotoxins, was cytolytic up to an equivalent of approximately 10(9 )Dt. The cytolytic agent(s) within the Bti/Btk-vegetative cell exoprotein (VCP) pool is an early spore outgrowth product identical to that of B. cereus and acting possibly by arresting protein synthesis. No cytolytic effects were seen with VCP from B. subtilis and Escherichia coli. These data, including recent epidemiologic work indicate that spore-containing BT products have an inherent capacity to lyse human cells in free and interactive forms and may also act as immune sensitizers. To critically impact at the whole body level, the exposure outcome would have to be an uncontrolled infection arising from intake of Btk/Bti spores. For humans, such a condition would be rare, arising possibly in equally rare exposure scenarios involving large doses of spores and individuals with weak or impaired microbe-clearance capacities and/or immune response systems.

Immune responses in farm workers after exposure to Bacillus thuringiensis pesticides.

Although health risks to pesticides containing Bacillus thuringiensis (Bt) have been minimal, the potential allergenicity of these organisms has not been evaluated. Therefore, a health survey was conducted in farm workers before and after exposure to Bt pesticides. Farm workers who picked vegetables that required Bt pesticide spraying were evaluated before the initial spraying operation (n = 48) and 1 and 4 months after (n = 32 and 20, respectively). Two groups of low- (n = 44) and medium- (n = 34) exposure workers not directly exposed to Bt spraying were also assessed. The investigation included questionnaires, nasal/mouth lavages, ventilatory function assessment, and skin tests to indigenous aeroallergens and to a variety of Bt spore and vegetative preparations. To authenticate exposure to the organism present in the commercial preparation, isolates from lavage specimens were tested for Bt genes by DNA-DNA hybridization. Humoral immunoglobulin G (IgG) and immunoglobulin E (IgE) antibody responses to spore and vegetative Bt extracts were assayed. There was no evidence of occupationally related respiratory symptoms. Positive skin-prick tests to several spore extracts were seen chiefly in exposed workers. In particular, there was a significant (p < 0.05) increase in the number of positive skin tests to spore extracts 1 and 4 months after exposure to Bt spray. The number of positive skin test responses was also significantly higher in high (p < 0.05) than in low- or medium-exposure workers. The majority of nasal lavage cultures from exposed workers was positive for the commercial Bt organism, as demonstrated by specific molecular genetic probes. Specific IgE antibodies were present in more high-exposure workers (p < 0.05) than in the low and medium groups. Specific IgG antibodies occurred more in the high (p < 0.05) than in the low-exposure group. Specific IgG and IgE antibodies to vegetative organisms were present in all groups of workers. Exposure to Bt sprays may lead to allergic skin sensitization and induction of IgE and IgG antibodies, or both.

Distribution of intercalative dye binding sites in chromatin.

Actinomycin D (AMD) and ethidium bromide (EB) were found to bind to chromatin isolated from a variety of gander tissues according to a strong and weak process analogous to that found for deproteinized DNA. Distribution of the dye intercalation sites in chromatin and DNA were evaluated at low r-values (dye bound per nucleotide) by following the appearance of free dye released from chromatin and DNA during thermal denaturation. The AMD dissociation profiles closely resembled the DNA or chromatin-DNA denaturation profiles; whereas the EB derivative dissociation profiles, indicated 3 major transitions for transcriptionally active chromatin with the main component corresponding to the single component which characterizes DNA. The DNA-like component was greatly reduced for mature erythrocyte chromatin but could be generated by removal of histone I and V. Removal of residual non acid-soluble proteins from dehistonized chromatin, urea treatment or dissociation and reconstitution of chromatin favoured conversion to the DNA-like component with loss of the other two. This study indicates that more than one type of binding exists generally in chromatin.

Glucanase gene diversity in prokaryotic and eukaryotic organisms.

A number of bacteria and eukaryotes produce extracellular enzymes that degrade various types of polysaccharides including the glucans starch, cellulose and hemicellulose (xylan). The similarities in the modes of expression and specificity of enzyme classes, such as amylase, cellulose and xylanase, suggest common genetic origins for particular activities. Our determination of the extent of similarity between these glucanases suggests that such data may be of very limited use in describing the early evolution of these proteins. The great diversity of these proteins does allow identification of their most highly conserved (and presumably functionally important) regions.

Fractionation of avian erythrocyte histones by hydrophobic interaction chromatography.

The interactions of H1 (H1A, H1B), H2A, H2B, H3, H4, and H5 with phenyl cross-linked agarose were studied. Procedures are described whereby all six histones can be bound, released, and fractionated by using appropriate salt concentrations or pH. The binding can be totally abolished by inclusion of hydrophobic disrupting agents. Control experiments with nonderivated cross-linked agarose ruled out a passive aggregation-disaggregation phenomenon governing the binding patterns. The absorption sequence based on the identification and quantitation of individual histones from either unfractionated (whole) histone or separate histone classes is as follows: H3 greater than or equal to H4 greater than H2B greater than or equal to H5 greater than or equal to H2A greater than H1A greater than or equal to H1B. The order differs only slightly from the reverse of the desorption sequence, H1B less than or equal to H1A less than or equal to H5 less than H2A less than or equal to H3. Preferential interaction of H2A-H2B, H3-H4, and H2A-H2B-H4 occur; these interactions can modify the original relative affinity of each individual component for the matrix. The variability in matrix affinity appears to involve simple stoichiometry of the histone components.

Multiplicity in cellulases of Schizophyllum commune. Derivation partly from heterogeneity in transcription and glycosylation.

The white-rot fungus, Schizophyllum commune, secretes a member of each of three classes of cellulases: a beta-glucosidase, an exoglucanase, and an endoglucanase. Antibodies were developed to members of each of these three enzyme classes. Secretion of these cellulases is induced when a mycelium is transferred from a glucose to cellulose medium. The maximum level of cellulase transcripts, as indicated by the ability to direct biosynthesis of these cellulases in the rabbit reticulocyte cell-free translation system, occurred when the rate of secretion was maximum. This implied that initial regulation, at least, of cellulase biosynthesis occurs at the transcriptional level. There were two distinct mRNA-directed products for each of the cellulases, with sizes estimated to be, for the beta-glucosidase, 95700 and 93800, for the exoglucanase, 59300 and 58200, and for the carboxymethylcellulase, 40600 and 39400. The secreted cellulases are largely glycosylated, as indicated by their binding to concanavalin A and their incorporation of D-[3H]mannose. The labelled protein was fractionated on concanavalin-A-agarose; about 70% of the label was bound. A small amount of each of the cellulases appeared in the unbound fraction; the remainder appeared in fractions eluted with 10 mM methyl glucoside or with 100 mM methyl glucoside plus 500 mM methyl mannoside. These results indicated each of the cellulases had an additional heterogeneity in glycosylation, with the most heavily glycosylated and highest molecular weight form eluting last from the concanavalin-A-agarose. Although tunicamycin (5 micrograms/ml) blocked glycosylation, there was still some secretion but at a reduced rate which was more pronounced for the beta-glucosidase than for the carboxymethylcellulase activity. The size of the tunicamycin-secreted product in each case was, within experimental error, equivalent to that of the mRNA-directed one.