Targeted mutagenesis of DNA with alkylating RecA assisted oligonucleotides.

Site-specific mutation was demonstrated in a shuttle vector system using nitrogen mustard-conjugated oligodeoxyribonucleotides (ODNs). Plasmid DNA was modified in vitro by ODNs containing all four DNA bases in the presence of Escherichia coli RecA protein. Up to 50% of plasmid molecules were alkylated in the targeted region of the supF gene and mutations resulted upon replication in mammalian cells. ODNs conjugated with either two chlorambucil moieties or a novel tetrafunctional mustard caused interstrand crosslinks in the target DNA and were more mutagenic than ODNs that caused only monoadducts.

Triplex targeting of a native gene in permeabilized intact cells: covalent modification of the gene for the chemokine receptor CCR5.

A 12 nucleotide oligodeoxyribopurine tract in the gene for the chemokine receptor CCR5 has been targeted and covalently modified in intact cells by a 12mer triplex forming oligonucleotide (TFO) bearing a reactive group. A nitrogen mustard placed on the 5'-end of the purine motif TFO modified a guanine on the DNA target with high efficiency and selectivity. A new use of a guanine analog in these TFOs significantly enhanced triplex formation and efficiency of modification, as did the use of the triplex-stabilizing intercalator coralyne. This site-directed modification of a native chromosomal gene in intact human cells under conditions where many limitations of triplex formation have been partially addressed underscores the potential of this approach for gene control via site-directed mutagenesis.

Sequence-specific targeting and covalent modification of human genomic DNA.

We compare two techniques which enable selective, nucleotide-specific covalent modification of human genomic DNA, as assayed by quantitative ligation- mediated PCR. In the first, a purine motif triplex-forming oligonucleotide with a terminally appended chlorambucil was shown to label a target guanine residue adjacent to its binding site in 80% efficiency at 0.5 microM. Efficiency was higher in the presence of the triplex-stabilizing intercalator coralyne. In the second method, an oligonucleotide targeting a site containing all four bases and bearing chlorambucil on an interior base was shown to efficiently react with a specific nucleotide in the target sequence. The targeted sequence in these cases was in the DQbeta1*0302 allele of the MHC II locus.

Modulation of glycosaminoglycan addition in naturally expressed and recombinant human thrombomodulin.

The two major glycoforms of full-length human thrombomodulin (TM), one with (TM(CS+)) and one without (TM(CS-)) chondroitin sulfate (CS) were analyzed on Western blots of primary and transformed cells and in cells expressing recombinant TM. TM on the surface of Chinese hamster ovary and COS-7 cells is solely TM(CS-). Primary arterial endothelial cells (HAEC and HPAEC) express a greater fraction of TM with CS attached than venous cells (HUVEC). Human lung carcinoma cells (A549) express more TM(CS+) than primary cells and recombinant TM on human melanoma cells (CHL-1) occurs in two very high molecular weight forms of TM(CS+). We explored this variation in TM(CS+) with soluble recombinant TM in several cell lines and analyzed the ambiguous CS addition site in human TM by site-directed mutagenesis. Mutation of Ser474 to Ala blocks CS addition in Chinese hamster ovary and COS-7 cells but not CHL-1 cells which add CS to Ser472 and Ser474. Structure of the O-link domain affects partitioning into TM(CS+) since substituting with the decorin CS addition sequence, substituting all Ser and Thr except Ser474 with Ala, and deleting around the potential beta-turn all increase the ratio of TM(CS+) to TM(CS-). A combination of the decorin substitution and deletion of the remaining O-link domain yields the most TM(CS+).

The gene for protein S maps near the centromere of human chromosome 3.

Two different mapping approaches were used to determine the human chromosomal location of the gene for protein S. A human protein S cDNA was used as a hybridization probe to analyze a panel of somatic cell hybrids containing different human chromosomes. Cosegregation of protein S-specific DNA restriction fragments with human chromosome 3 was observed. Three cell hybrids containing only a portion of chromosome 3 were analyzed in order to further localize protein S. Based on the somatic cell hybrid analysis, protein S is assigned to a region of chromosome 3 that contains a small part of the long arm and short arm of the chromosome including the centromere (3p21----3q21). In situ hybridization of the protein S cDNA probe to human metaphase chromosomes permitted a precise localization of protein S to the region of chromosome 3 immediately surrounding the centromere (3p11.1----3q11.2). Protein S is the first protein involved in blood coagulation that has been mapped to human chromosome 3.

Characterization of cDNA and genomic sequences corresponding to an embryonic myosin heavy chain.

We report here the isolation and characterization of cDNA and genomic sequences corresponding to a rat embryonic myosin heavy chain (MHC) protein. This gene, which is present as a single copy in the rat genome, comprises about 25 kilobase pairs of DNA and contains approximately 80% intronic sequences. The embryonic MHC gene belongs to a highly conserved multigene family, and exhibits a high degree of nucleotide and amino acid sequence conservation with other sarcomeric MHC genes from nematode to man. S1 nuclease mapping experiments using cDNA and genomic probes show that this MHC gene is transiently expressed during skeletal muscle development. Its mRNA is detected in fetal skeletal muscle during early development and persists up to 2 weeks after birth with the overlapping expression of neonatal and adult skeletal MHC mRNAs. However, this MHC is not expressed in the adult skeletal muscle with the exception of extraocular muscle fibers. The transient expression during muscle development of the isoform produced by this gene and its sequential replacement by other MHCs raises interesting questions about the mechanism controlling MHC isozyme transitions and the physiological significance of the individual MHCs in muscle fibers.

Characterization of sarcomeric myosin heavy chain genes.

Myosin heavy chain is encoded by a large multigene family. Using pMHC-25, a recombinant cDNA clone isolated from the rat myogenic cell line L6E9, four members of this family in the rat have been isolated and shown to be tissue-specific and developmentally regulated. The coding regions of these genes share regions of homology interspaced with regions of non-homology. Detailed analysis of one embryonic and one adult myosin heavy chain gene shows that the coding sequences are interrupted by numerous intervening sequences whose number, size, and distribution do not appear to be conserved in the same organism or between species.

Small RNA molecules related to the Alu family of repetitive DNA sequences.

A rodent 4.5S RNA molecule with extensive homology to the Alu family of interspersed repetitive DNA sequences has been found physically associated with polyadenylated nuclear and cytoplasmic RNAs (W. Jelinek and L. Leinwand, Cell 15:205-214, 1978; S. Haynes et al., Mol. Cell. Biol. 1:573-583, 1981). In this report, we describe a 4.5S RNA molecule in rat cells whose RNase fingerprints are identical to those of the equivalent mouse molecule. We show that the rat 4.5S RNA is part of a small family of RNA molecules, all sharing sequence homology to the Alu family of DNA sequences. These RNAs are synthesized by RNA polymerase III and are developmentally regulated and short-lived in the cytoplasm. Of this family of small RNAs, only the 4.5S RNA is found associated with polyadenylated RNA.

Sarcomeric myosin heavy chain is coded by a highly conserved multigene family.

pMHC25, a recombinant plasmid containing myosin heavy chain (MHC) cDNA sequences from differentiated myotubes of the L6E9 rat cell line, has been shown to hybridize to all sarcomeric MHC mRNAs so far tested but not to nonsarcomeric MHC mRNAs. In addition, pMHC25 hybridizes to multiple restriction endonuclease-digested fragments of rat genomic DNA corresponding to different MHC genomic sequences. Thus, the MHC gene represented by pMHC25 is a member of a sarcomeric MHC multigene family that has regions of sequence homology shared among its members. This sarcomeric MHC multigene family has been estimated to be composed of a minimum of seven genes, some of which are polymorphic in the rat. We have also determined that pMHC25 hybridizes to MHC gene sequences in genomic DNA of all species that have striated muscle, ranging from nematodes to man. Sarcomeric MHC genes, therefore, have been horizontally and vertically conserved in evolution. Additionally, we have used the pMHC25 plasmid to demonstrate that MHC genes do not undergo rearrangement or amplification during muscle cell differentiation.

Moloney murine sarcoma proviral DNA is a transcriptional unit.

A portion of Moloney murine sarcoma virus DNA which is repeated at both ends of the provirus has been sequences. The nucleotide sequence, together with hybridization data obtained with in vitro pulse-labelled nascent viral RNA, indicate that initiation and termination of RNA synthesis occur within that region of the proviral DNA. A model for transcriptional readthrough of termination signals during RNA synthesis in this system is suggested.

Cytoplasmic processing of myosin heavy chain messenger RNA: evidence provided by using a recombinant DNA plasmid.

A recombinant DNA plasmid, designated pMHC25, has been constructed that contains structural gene sequences for rat skeletal muscle myosin heavy chain (MHC). The identity of the MHC sequence insert in pMHC25 was determined by muscle-tissue specificity, inhibition of MHC protein synthesis in vitro by hybrid-arrested translation, purification of mRNA that directs the synthesis of MHC protein in vitro, and hybridization to a 33S cytoplasmic mRNA found only in differentiated muscle cells. pMHC25-DNA-excess filter hybridizations were used to show that more than 90% of the newly synthesized MHC mRNA that appears in the cytoplasm of differentiated L6E9 myotubes contains a long 3' poly(A) tail. In contrast, 90% of the MHC mRNA that accumulates in the cytoplasm of these same cells during myogenic differentiation lacks this long 3' poly(A) tail. These results suggest the occurrence of a posttranscriptional event in differentiated L6E9 myotubes that involves the cytoplasmic processing of poly(A)+ MHC mRNA to poly(A)- or poly(A)-short MHC mRNA.

Structure of functional A salina -- E coli hybrid ribosome by electron microscopy.

Small 40S Artemia salina and large 50S Escherichia coli ribosomal subunits can be assembled into 73S hybrid monosomes active in model assays for protein synthesis. The reciprocal combination -- small 30S E coli and large 60S A salina -- fails to form hybrids. The 73S hybrid particles strongly resemble homologous 70S E coli and 80S A salina monosomes. The morphologic differences between the corresponding eukaryotic and prokaryotic ribosomal particles, established by electron microscopy, do not significantly affect the assembly and mutual orientation of 40S A slina and 50S E coli subunits in the heterologous monosome. The fact that the structure of the interface, the supposed site of protein synthesis, is preserved in the active hybrid implies that retention or loss of biologic activity of hybrid ribosomes is determined by the extent of conformational changes in the interface.

Cell-bound cations of the moderately halophilic bacterium Vibrio costicola.

Over most of the range of salt concentrations in which the moderately halophilic bacterium Vibrio costicola could grow, the sum of the cell-associated Na+ + K+ ions was at least as high as in the external medium. This is in contrast to other moderate halophiles, which have substantially lower internal than external salt concentrations for most of their growth range. The relative amounts of Na+ and K+ in V. costicola varied with environmental conditions. The K+/Na+ ratio fell during anaerobic incubation or when cells were poisoned. As Na+ ions left the cells, K+ ions entered. However, movement of these ions was not tightly coupled, since K+ content of cells could increase without a corresponding decrease in Na+ content. The Mg2+ contents of cells varied little with environmental conditions.