Annie C. Y. Chang

The organization of sea urchin histone genes

Sucrose gradient analysis of total sea urchin DNA cleaved with the EcoRI and Hind III restriction endonucleases and identification of histone coding gene sequences by hybridization with histone mRNA have elucidated the basic organization of the histone gene repeat unit. These data, plus results obtained by electrophoretic analysis of purified endonuclease-cleaved sea urchin histone DNA and hybridization with cRNA transcribed from the eucaryotic segment of constructed plasmid chimeras cloned in E. coli, show that the several DNA sequences coding for individual histone proteins are intermingled in a 7 kilobase (kb) repeat unit. Cleavage of total sea urchin DNA with EcoRI produces 2.2 and 4.8 kb fragments, and which are contained in a 7 kb Hind III fragment. Cleavage with both enzymes reveals that the 2.2 kb EcoRI fragment contains a Hind III site 0.15--0.2 kb from an end. RNA.DNA hybridization between chimeric palsmic DNA and purified individual mRNAs isolated from sea urchin embryo polyribosomes has been used to assign coding sequences to either the 2.2 or 4.8 kb region of the histone DNA repeat unit. A map of the histone genes is proposed.

Genetic expression in bacteriophage λ: III. Inhibition of Escherichia coli nucleic acid and protein synthesis during λ development☆☆☆

Abstract The effects of λ phage development on the synthesis of Escherichia coli DNA, RNA and protein have been studied using incorporation of radioactively labeled precursors and host β-galactosidase production as measurements of bacterial macromolecular metabolism. The ability of certain λ mutants to affect host functions was examined, and DNA-DNA or DNA-RNA hybridization served to distinguish bacterial from viral nucleic acids. The results of these experiments indicate that: (1) major inhibition of bacterial RNA and protein synthesis occurs following either λ phage infection or induction of a lysogen by heat or mitomycin C; shut-off of host DNA synthesis by λ appears to be less prompt and less prominent; (2) depression of host function results from induction of a prophage situated trans to the chromosome on the episome F′- gal , indicating that a diffusable phage gene product is involved; (3) synthesis of new protein is implicated in the phage-directed alteration of bacterial metabolism; and (4) the ability to depress bacterial functions has been localized to a segment of the phage chromosome near the genes producing exonuclease and β-protein.

A method for selective cloning of eukaryotic DNA fragments in Escherichia coli by repeated transformation

SummaryA procedure is described for the selective cloning in E. coli of restriction endonuclease-generated fragments of eukaryotic DNA linked to a bacterial plasmid replicon. The cloning method involves the initial use of tranformation to amplify an unfractionated heterogeneous population of ligated DNA molecules, and the subsequent purification of plasmid chimeras by repeated cycles of transformation and gradient centrifugation. The procedure has enabled the identification and purification of a rare 6.8x106 dalton EcoRI-generated fragment of X. laevis rDNA that is present in about 0.5% of the rDNA tandem repeat units.

Phenotype-Based Identification of Host Genes Required for Replication of African Swine Fever Virus

ABSTRACT African swine fever virus (ASFV) produces a fatal acute hemorrhagic fever in domesticated pigs that potentially is a worldwide economic threat. Using an expressed sequence tag (EST) library-based antisense method of random gene inactivation and a phenotypic screen for limitation of ASFV replication in cultured human cells, we identified six host genes whose cellular functions are required by ASFV. These included three loci, BAT3 (HLA-B-associated transcript 3), C1qTNF (C1q and tumor necrosis factor-related protein 6), and TOM40 (translocase of outer mitochondrial membrane 40), for which antisense expression from a tetracycline-regulated promoter resulted in reversible inhibition of ASFV production by >99%. The effects of antisense transcription of the BAT3 EST and also of expression in the sense orientation of this EST, which encodes amino acid residues 450 to 518 of the mature BAT3 protein, were investigated more extensively. Sense expression of the BAT3 peptide, which appears to reversibly interfere with BAT3 function by a dominant negative mechanism, resulted in decreased synthesis of viral DNA and proteins early after ASFV infection, altered transcription of apoptosis-related genes as determined by cDNA microarray analysis, and increased cellular sensitivity to staurosporine-induced apoptosis. Antisense transcription of BAT3 reduced ASFV production without affecting abundance of the virus macromolecules we assayed. Our results, which demonstrate the utility of EST-based functional screens for the detection of host genes exploited by pathogenic viruses, reveal a novel collection of cellular genes previously not known to be required for ASFV infection.

Sequence of the cDNA encoding porcine pro-opiomelanocortin

Messenger RNA was extracted from porcine pituitary pars intermedia and cloned as cDNA by standard methods. The nucleotide sequence encoding porcine pro-opiomelanocortin was established from analysis of two separate cDNA segments having an overlap of 420 bases. The amino acid sequence for the porcine pro-opiomelanocortin protein, which was inferred from the cDNA sequence, was found to correspond exactly to the sequence determined by direct amino acid analysis of the component peptides of pro-opiomelanocortin; namely, the porcine hormones ACTH, beta-lipotropin, gamma-MSH and the connecting peptide. We thus find no evidence for the existence of two porcine pro-opiomelanocortin genes that differ in their coding sequences, as was suggested by the in vitro protein synthesis results of others using mRNA obtained from porcine pituitaries.

Genetic expression in bacteriophage λ: IV. Effects of P2 prophage on λ inhibition of host synthesis and λ gene expression

The effects of P2 prophage on lambda-mediated depression of host metabolism, and on lambda macromolecular synthesis have been studied. Experiments employing certain lambdabio deletion mutants and lambda-related phages suggest that the same gene products are involved in inhibition of host metabolism (Hin+ function) and susceptibility to P2 inhibition (Spi+ function). Synthesis of early lambda mRNA and protein occurs normally during the first 10 min after lambda infection of P2 lysogens, but subsequent expression of early and late lambda genes is interfered with by the combined actions of the P2 old gene and lambda Hin+ (Spi+) function. In addition, the P2 old+ product enhances the inhibitory effects of lambda Hin+ function on host metabolism, and can substitute for a component of this function lacking in lambdabio 72 and lambdabio 69.

STUDIES OF CLONED DNA ENCODING THE STRUCTURE FOR THE BOVINE CORTICOTROPIN‐ß‐LIPOTROPIN PRECURSOR PROTEIN*

The pituitary hormones corticotropin (ACTH) and /Mipotropin (IJLPH) are formed from a large common precursor protein.’-’ These two hormones in turn are known to contain small component peptides having biological activity: a-melanotropin (aMSH) and corticotropin-like intermediate lobe peptide (CLIP) are derived from ACTH.’ /3-melanotropin (IJMSH), endorphins, and methionine enkephalin are liberated from /lLPH.9-’’ The locations of these various peptide components of the precursor protein are shown in FIGURE 1. The intracellular level of the messenger RNA (mRNA) encoding the common precursor protein is depressed by glucocorticoids, which appear to act at the transcriptional level by means of the glucocorticoid receptor. 1 2 . 1 3 Although the general positions of ACTH and PLPH on the common precursor molecule have been known for several years, earlier studies had provided no information about the exact structural relationships of these peptides. Moreover, ACTH and PLPH account for only one-third to one-half the molecular weight of the precursor protein and thus, there has been considerable interest in and speculation about primary structure and possible biological functions of the peptides in the remaining (“cryptic”) portion. together provide a powerful tool for understanding the organization of eukaryotic genes and the regulation of their expression. Using bacterial plasmids or phage vectors, it has been possible to propagate DNA sequences from a wide variety of plant and animal cell sources within bacterial cells (for reviews, see references 17-19). Procedures for the introduction of randomly generated fragments of mammalian chromosomal DNA into Escherichia coli K12,’O for the cloning of complementary DNA (cDNA) copies of RNA species that predominate in certain and for the propagation in bacteria of chemically synthesized nucleotide chains that correspond to the amino acid sequence of small peptide hormone~~‘.~’ have been described. Recently, we reported the cloning and analysis of a double-stranded cDNA The techniques of DNA cloning“ and nucleotide sequence

Replication and Expression of Constructed Plasmid Chimeras in Transformed Escherichia coli—A Review

EcoRI restriction-endonuclease-generated fragments of bacterial plasmids isolated from Staphylococcus aureus or Escherichia coli, or of amplified DNA coding for the 18S and 28S ribosomal RNA of Xenopus laevis, have been linked to the pSC101 plasmid replicon and introduced into E. coli by transformation. The constructed plasmid chimeras can be cloned as stable replicons in E. coli, where they synthesize RNA and/or protein products specified by their component genes.