Presence of DNA polymerase in lymphosarcoma in northern pike (Esox lucius).

Northern poke lymphosarcoma DNA polymerase was partially purified from particulate fractions banding at 1.15 to 1.16 g/ml from homogenates prepared from frozen necropsies of tumor-bearing pike. The enzyme behaves as a typical reverse transcriptase, in that it prefers ribotemplates to deoxytemplates. The isoelectric point (pl 5.5) is similar to that of avian myeloblastosis virus polymerase. The pike enzyme elutes from a phosphocellulose column at 0.22 M potassium phosphate, the same as avian myeloblastosis virus DNA polymerase. The enzyme activity is inhibited by pyran, a specific inhibitor of viral DNA polymerases. The most striking difference between the pike lymphoma polymerase and the other viral DNA polymerases tested is the low maximum temperature of 20 degrees, compared to 30 degrees for Rauscher leukemia virus polymerase and 38 degrees for avian myeloblastosis virus and Rous sarcoma virus.

Cloning and sequence analysis of an H(+)-ATPase-encoding gene from the human dimorphic pathogen Histoplasma capsulatum.

A gene related to the PMA1 gene from Saccharomyces cerevisiae was isolated from the pathogenic human dimorphic fungus, Histoplasma capsulatum, using fungal-specific oligodeoxyribonucleotide (oligo) probes. This gene has been given the name Hc-PMA1. The structural organization of Hc-PMA1 consists of three exons (375, 2329 and 44 bp) and two introns (115 and 116 bp). The nucleotide sequence predicts an H(+)-ATPase-related protein of 916 amino acids (aa). Comparison of the deduced aa sequence to that of Neurospora crassa and S. cerevisiae (PMA1) plasma membrane H(+)-ATPases showed a greater similarity to that from N. crassa (85% identity). Furthermore, the two introns in the Hc-PMA1 gene interrupt the coding region in the precise locations determined for two of the four N. crassa Nc-PMA introns. H. capsulatum intron 1 contains two repeat motifs, d(TA)16 and d(TG)10, each potentially capable of forming non-B DNA structures. Northern analysis of H. capsulatum total RNA indicated that the Hc-PMA1-specific mRNA is approx. 3.3 kb in size, in agreement with the predicted size of the gene.

The inhibition of Rauscher leukemia virus and avian myeloblastosis virus DNA polymerases by anthracycline compounds.

Studies by other investigators have shown that adriamycin and daunorubicin exhibit antitumor and antiviral activity. A possible antiviral mechanism for the anthracycline compounds is the potent inhibition of viral DNA polymerases. Five anthracycline compounds were tested against purified Rauscher leukemia virus and avian myeloblastosis virus DNA polymerases. All compounds were found to be potent inhibitors of viral DNA polymerase activity. Inhibition was found to be primarily due to the planar ring structure (daunomycinone) common to all of these compounds. The degree of inhibition was dependent on the templates used: activated DNA, synthetic hybrids, poly(rA).dT12-18 and poly(rC).dG12-18, and the synthetic copolymer, poly(DA-dT). Alteration of the group substituent on the planar ring affected the degree of viral DNA polymerase inhibition. The inhibitory effects by anthracycline compounds appear to be relatively specific for viral polymerases.

Rat intermediate lobe in culture: dopaminergic regulation of POMC biosynthesis and cell proliferation.

The effects of the selective D-2 selective agonist, quinpirole, on biosynthesis of proopiomelanocortin (POMC) and cell proliferation rate of cultures of rat intermediate lobe (IL) have been examined. Primary cultures of rat IL were prepared by mechanically dispersing IL lobules in medium. Following a six day incubation, approximately 25% of the cells settled onto the culture plate and began to extend into a monolayer. Quinpirole markedly reduced immunoreactive beta-endorphin levels in the medium and POMC mRNA in both the attached and floating lobules. The incorporation of 35S-methionine into 32 kDa POMC, a 18-22 kDa complex of proteins, a 16 kDa protein and a 15 kDa protein was decreased significantly in both the attached and floating lobules. In contrast, the proliferation in the floating, but not the attached, cells was inhibited by quinpirole. The floating IL lobule appears to provide a reasonably faithful model of the dopaminergic regulation of IL function in vivo, while the attached IL cells may provide an interesting tool to study the regulation of IL cell proliferation.

Direct detection of Histoplasma capsulatum in soil suspensions by two-stage PCR.

Histoplasmosis is the most common pulmonary mycosis in the United States. The responsible fungal pathogen, Histoplasma capsulatum, grows in soils contaminated with bird or bat droppings. Inhalation of dust from contaminated areas containing H. capsulatum spores is a primary route of infection. The ability to detect H. capsulatum in soil samples has been limited by the lack of fast, reliable and inexpensive methods. A polymerase chain reaction (PCR) method was developed that allows the direct detection of H. capsulatum in soil. A two-stage PCR protocol was followed employing both fungal-specific primers and nested primers specific for the internal transcribed spacer (ITS) region of the 5.8S rRNA gene of H. capsulatum. The estimated limit of detection of this method is 10 spores. In contrast to the more expensive and indirect mouse inoculum assay, which requires 6-8 weeks for sample analysis, PCR analysis of soil contaminated with H. capsulatum can be completed in less than 2 days.

In vivo and in vitro complementation between guaB and in vivo complementation between guaA auxotrophs of Salmonella typhimurium.

guaA and guaB mutants of Salmonella typhimurium were isolated utilizing the mutagen, nitrous acid. The guaB mutants were defective for inosine 5'-monophosphate (IMP) dehydrogenase activity and those mutants classified as guaA exhibited no xanthosine 5'-monophosphate aminase activity. In vivo complementation maps were determined for the mutants. The guaB map indicated that at least three complementation regions existed whereas five complementation regions were observed for the guaA mutants. The demonstration of in vitro complementation was also achieved for the guaB mutants by utilizing a process of denaturation and renaturation. All of the guaB mutants that exhibited in vivo complementation were found to exhibit in vitro complementation. No correlation was found between the degree of in vivo complementation exhibited by the various pairs of mutants and the specific enzyme activities of the same mutant pair that yielded in vitro complementation. The kinetic parameters for three of the most active guaB "complemented" enzymes and a renatured wild-type IMP dehydrogenase were then examined. No apparent differences were found in the K(m) values between any of the enzymes for substrate, IMP, activator ion, K(+), and coenzyme, oxidized nicotinamide adenine dinucleotide. Some differences were noted in the apparent V(max) values; the best "complemented" enzyme yielded only 20% of the velocity exhibited by renatured wild-type enzyme.

Sampling and analytical method development for qualitative assessment of airborne mycobacterial species of the Mycobacterium tuberculosis complex.

This article presents a novel, qualitative approach for detecting airborne M. tuberculosis. Culturing or sample purification is not required. A DNA diagnostic method involving the polymerase chain reaction (PCR) coupled to an enzymatically generated color reaction was used for direct detection of M. bovis BCG (Bacillus of Calmette-Guérin), a surrogate for pathogenic M. tuberculosis. Fewer than 10 mycobacteria were detected with no culturing using this bioanalytical method. Analysis was completed in 1 to 1.5 days, in contrast to traditional culturing methods requiring a minimum of 2-3 weeks. To evaluate an air sampling method coupled to a PCR bioanalytical method, liquid cultures of the surrogate were aerosolized and collected for PCR analyses using 37-mm filter cassettes containing polytetrafluoroethylene filters. An Andersen six-stage (viable) particle sizing sampler was employed as a reference sampler. Aerosolized BCG impacted onto Andersen agar plates required incubation periods of 6-8 weeks before small colony forming units could be detected and enumerated. Although the BCG mean length of the rod-shaped particles was 8.3 microns, the airborne BCG particles were collected predominantly on the Andersen 4-6 stages, representing aerodynamic diameters 0.7 to 3.3 microns. Approximately 25 mycobacteria were detected without culturing using the PCR-filter cassette method. This approach could be used to detect airborne mycobacterial species of the M. tuberculosis complex and could permit the early detection of contaminated indoor air. Also, the efficacy of environmental controls could be evaluated and monitored. This approach could also be used to study the expulsion of infectious particles from patients and may permit risk assessment in regard to personal respiratory protection.

Localization of neuropeptide Y messenger ribonucleic acid in rat and mouse brain by in situ hybridization.

The distribution of neuropeptide Y (NPY) messenger ribonucleic acid (mRNA) in the rat and mouse brain has been examined. A 28-mer oligonucleotide probe complementary to a distinctive region of NPY mRNA was 3'-end labeled with [35S]-deoxycytosine triphosphate ([35S]-dCTP) by using terminal transferase and was hybridized to fixed sections of rat and mouse brain. The hybridization of the labeled probe to the mRNA in the tissue section was then detected autoradiographically. A strong hybridization signal was seen corresponding to the arcuate nucleus of the hypothalamus. Scattered NPY-mRNA-containing cell bodies were seen throughout the cerebral cortex. Experiments which compared the distribution of NPY mRNA with immunohistochemically detected NPY indicated a similar distribution in the arcuate nucleus of the hypothalamus and cerebral cortex. These results indicate that active transcription of the NPY gene occurs within the brain and this transcription takes place in NPY-containing cell bodies.

Compensatory increase in levels of beta minor globin in murine beta-thalassemia is under translational control.

A 3.7-kilobase pair deletion including the entire beta major globin gene results in beta-thalassemia in a murine model of the disease (Skow, L. C., Burkhart, B. A., Johnson, F. M., Popp, R. A., Popp, D. M., Goldberg, S. Z., Anderson, W. F., Barnett, L. B., and Lewis, S. E. (1983) Cell 34, 1043-1052). There is a compensatory increase in synthesis of beta minor globin, resulting in a beta minor/alpha globin ratio of 0.75 in the homozygous thalassemic mouse, as compared to 0.2 in the normal homozygous diffuse mouse. The results presented here demonstrate that the increase in beta minor globin synthesis occurs at translation rather than at transcription. RNase T1 analysis of reticulocyte mRNA reveals that the beta/alpha ratio of globin mRNA is 0.3, significantly lower than the globin synthetic ratio of 0.7. However, the beta/alpha ratio of mRNA on polysomes is higher than unassociated mRNA, demonstrating that beta minor mRNA is preferentially translated. Elevated synthesis of beta minor globin is maintained during in vitro translation in thalassemic reticulocyte lysate. In this system, partial inhibition of translational elongation by cycloheximide decreases the beta minor/alpha globin synthetic ratio, whereas partial inhibition of initiation by hemin deficiency increases the beta minor/alpha synthetic ratio. This suggests that beta minor mRNA competes with alpha-mRNA for a limiting mRNA binding factor at initiation of translation.

Structural organization of a 17 KB segment of the alpha 2 collagen gene: evaluation by R loop mapping.

A recombinant phage, SpC3, containing a 17 kb genomic DNA insert representing approximately 60% of the 3' portion of the sheep collagen alpha 2 gene, was evaluated by electron microscopic R loop analysis. A minimum of 17 intervening sequences (introns) and 18 alpha 2 coding sequences (exons) were mapped. With the exception of the 850 base pair exon located at the extreme 3' end of the insert, all exons contained 250 base pairs or less. The total length of all the exons in SpC3 was 3,014 base pairs. The length distribution of the 17 introns ranged from 300 to 1600 base pairs; together, all of the introns comprised 14,070 base pairs of SpC3 DNA. Thus, the DNA region required for coding the interspersed 3 kb of alpha 2 collagen genetic information was 5.6 fold longer than the corresponding alpha 2 mRNA coding sequences.

The purification and characterization of subunits alpha, beta, and gamma from the rabbit reticulocyte eukaryotic initiation factor 2.

Eukaryotic initiation factor 2 (eIF-2) contains three nonidentical subunits, alpha, beta, and gamma. The simultaneous purification of all three subunits was achieved by reverse-phase HPLC using a 0.1% trifluoroacetic acid-acetonitrile binary solvent system. The order of the eluted subunits, beta, alpha, and gamma, was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After hydrolysis in 6 N HCl, picomole level amino acid composition analysis was achieved by the ninhydrin reaction on a Beckman 6300 system. Using second-derivative spectroscopic analysis, Trp was detected in all three subunits. All three subunits were subjected to amino-terminal sequence analysis. The amino-terminal of eIF-2 alpha from amino acid positions 1 to 23 inclusive was determined. The order of eight amino acids from the amino-terminal of eIF-2 gamma was also determined. This characterization and partial determination of the primary sequence of these subunits permit the utilization of molecular biology techniques in order to elucidate the complete primary structure. Additionally, the partial amino acid sequence data permitted the designation of synthetic gene probes as well as the identification of eIF-2 alpha and gamma cDNA and/or genomic clones.

Control of collagen production by human diploid lung fibroblasts.

The fibroblast is a differentiated mesenchymal cell which produces and exports collagen, a macromolecule that plays a critical structural role in the function of most organs. To evaluate the control soft tissue fibroblasts have over collagen production, HFL-1, a diploid human lung cell strain, was studied during periods of rapid cell growth and relatively slow growth over 25 population doublings. To minimize environmental influences, the extracellular milieu of the cells was kept constant throughout the study period. Rates of collagen production per cell per unit time were quantitated by labeling HFL-1 with [14C]proline and measuring the production of [14C]hydroxyproline after taking into consideration the specific activity of [14C]proline within the free intracelllular proline pool and the per cent hydroxylation of proline residues in newly synthesized collagen. Although the specific activity of intracellular free proline and the per cent hydroxylation of proline in collagen varied considerably depending on the growth rates of the cells, collagen production by HFL-1 was constant, even during periods of rapid cell growth. Thus, under conditions of a stable environment, populations of soft tissue fibroblasts rigidly control their collagen production. In cultures that maintained a constant doubling time, this stability was maintained over at least 25 population doublings, suggesting that on the average, collagen production appears to be tightly controlled and dissociated from the events and sequelae of cell division.

Sheep elastin genes. Isolation and preliminary characterization of a 9.9-kilobase genomic clone.

A sheep genomic library containing sheep DNA in the bacteriophage vector Charon 4A was screened for elastin-gene sequences with partially purified, 32P-labelled elastin mRNA (mRNAE). A recombinant containing a 9.9-kb (kilobase) insert was selected from several positive clones by secondary and tertiary screening for further characterization. Positive identification of this elastin clone, designated SE1, was made with radiolabelled mRNAE by hydridization-selected translation and Southern blotting of restriction-enzyme fragments of SE1 DNA. Hybridization of either mRNAE or elastin complementary DNA to restriction fragments of SE1 showed that most of these fragments of SE1 contained elastin-coding sequences. Orientation of the insert was established by preferential hybridization of a short complementary elastin DNA to restriction fragments adjacent to the right arm of Charon 4A. Reciprocal hybridizations of nick-translated SE1 and sheep genomic DNA on Southern blots showed that two restriction fragments of SE1 contained sequence elements which were repeated at high frequency in a restriction-endonuclease-EcoR1 digest of total sheep genomic DNA. In the accompanying paper [Davidson, Shibahara, Boyd, Mason, Tolstoshev & Crystal (1984) Biochem. J. 220, 653-663], it is shown that a subcloned fragment of this elastin gene quantitatively and specifically hybridized to mRNAE sequences in sheep tissue RNA. Electron microscopy of SE1-mRNAE hybrids indicated the presence of at least seven large R-loops. Measurements of these structures indicated that SE1 is likely to contain less than 2 kb of coding sequence and more than 8 kb of intervening sequence, with an average exon size of 120 base-pairs. Thus the elastin gene is distributed over an extended region of the sheep genome and contains numerous intervening and coding sequences.

Isolation and characterization of a 15-kilobase genomic sequence coding for part of the Pro alpha 2 chain of sheep type I collagen.

DNA fragments, prepared by partial Eco RI digestion of fetal sheep liver genomic DNA, were used to prepare a "library" of amplified genomic sequences with the lambda vector Charon 4A. Several recombinant plaques were identified by their ability to hybridize to 32P-labeled cDNA prepared from fetal sheep tendon type I procollagen mRNA. Two of these recombinant DNA bacteriophages (SpC3 and SpC7) were identified as containing procollagen pro alpha 2 gene sequences by their ability to specifically anneal to procollagen pro alpha 2 mRNA. Restriction endonuclease and hybridization to a cloned pro alpha 2 cDNA demonstrated that approximately half (2.5 kilobases) of the pro alpha 2 mRNA sequence is distributed over 15 kilobases of genomic DNA. Restriction maps of SpC3 and SpC7 demonstrated that these two DNA fragments contain overlapping sequences of the pro alpha 2 gene. Electron microscopy and R-loop analysis of SpC3 revealed that at least 12 to 16 intervening sequences are distributed throughout the length of this gene fragment.

A 66-base pair insert bridges the deletion responsible for a mouse model of beta-thalassemia.

The breakpoints of the deletion responsible for the Hbb(th-1) mouse model of beta-thalassemia have been isolated. A 3709 (+/- 2)-base pair (bp) region, including the entire beta major globin gene and 2 kilobases of 5' flanking region, is deleted. A novel 66 (+/- 2)-bp sequence, ending in a stretch of 25 dA:dT base pairs, was found to bridge the deletion. A region of the normal murine genome, containing the first 43 bp of the deletion-associated insert (DAI), but lacking the 25-bp dA:dT sequence, was isolated. All normal mice tested contain this DAI-like element and several inbred strains contain an additional DAI-like element. The sequence spanning the Hbb(th-1) deletion may be a reverse transcript of this region.

Organization of a type I keratin gene. Evidence for evolution of intermediate filaments from a common ancestral gene.

The genomic structure of the mouse 59-kDa keratin gene, a Type I intermediate filament (IF) gene is presented. A comparison of the organization of this gene with that of the human 67-kDa keratin, a Type II IF gene, and hamster vimentin, a Type III IF gene, suggests a common evolutionary origin for Type I, II, and III IF genes. Most introns in these three types of IF genes occur at similar positions within the region encoding sequences predicted to form coiled-coils, but do not delineate structural subdomains. Interestingly though, most of the introns interrupt at or near the beginning of the characteristic 7-residue (heptad) repeat of sequences which form the coiled-coil. These data suggest that the three types of IF genes arose from a common ancestor which may have been assembled from smaller units containing multiple heptad repeats. Subsequent duplication events may then have formed the three known alpha-helical types and each of their various members.

Isolation and cDNA sequence of human postheparin plasma hepatic triglyceride lipase.

Hepatic triglyceride lipase (H-TGL) was isolated from human postheparin plasma by column chromatography on heparin-Sepharose and phenyl-Sepharose and immunoaffinity chromatography with monoclonal antibodies. The purified enzyme had an apparent molecular weight of 65,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an amino-terminal sequence of Leu-Gly-Gln-Ser-Leu-Lys-Pro-Glu. Partial amino acid sequences of seven cyanogen bromide peptides were obtained. A human hepatoma cDNA library was screened with synthetic oligonucleotides derived from the partial protein sequence. The cloned H-TGL cDNA of 1569 nucleotides predicts a mature protein of 477 amino acids plus a leader sequence of 22 amino acids. Blot hybridization analysis of poly(A)+ mRNA with a putative H-TGL cDNA clone gave a single hybridizing band of 1.7 kilobases. The protein contains four consensus N-glycosylation sequences based on the cDNA sequence. Comparison of the enzyme sequence with that of other lipases reveals highly conserved sequences in regions of putative lipid and heparin binding. The carboxyl terminus of H-TGL contains a highly basic sequence which is not reported to be present in rat H-TGL or other members of the lipase gene family.