Jack G. Chirikjian

Function and properties of aminoacyl transferases and aminoacyl-tRNA synthetases in rat liver and HeLa cells.

Abstract Elongation factors 1 and 2, and aminoacyl-tRNA synthetases, are heterogeneously distributed in sucrose gradients containing postribosomal extracts of HeLa cells. The highest activity of the enzymes was found in a 5–7S region which corresponds to sedimentation coefficients expected for the free enzymes, but considerable enzyme activity was present at higher S values. The two elongation factors were found in a 10–17S region, whereas the synthetase activity was broadly distributed between 7 and 26S. The 26S peak contained mainly the aminoacyl-tRNA synthetases for Val, Ileu Leu, Lys, and His. In contrast, the synthetases for Met, Phe, and Ser were dominating in the 5–10S region of the gradients. Two regions of a sucrose gradient containing postribosomal cell extract were subjected to gel filtration on a calibrated Sepharose 6 B column. One of the samples contained enzymes with a sedimentation coefficient of 10–12S, and the other sample, serving as a control, contained free, noncomplexed enzymes. EF-2 and synthetases eluted from 10–12S material mainly as high molecular weight aggregates ( M r up to approx. 3 million), whereas the eluate of 5S material contained both enzymes in their free, nonaggregated forms. EF-1 and EF-2 activity was monitored during the purification of leucyl-tRNA synthetase from rat liver. At a stage where purification was 350-fold and the enzyme was essentially devoid of all other synthetase activities, significant EF-1 and EF-2 activity still remained with the enzyme. This complex from rat liver has an apparent molecular weight of 5 × 10 5 .

Mutation identification DNA analysis system (MIDAS) for detection of known mutations.

We introduce a novel experimental strategy for DNA mutation detection named the Mismatch Identification DNA Analysis System (MIDAS) [1, 2], which has an associated isothermal probe amplification step to increase target DNA detection sensitivity to attomole levels. MIDAS exploits DNA glycosylases to remove the sugar moiety on one strand (the probe strand) at a DNA base pair mismatch. The resulting apyrimidinic/apurinic (AP) site is cleaved by AP endonucleases/lyases either associated with the DNA glycosylase or externally added to the reaction mixture. MIDAS utilizes 32P‐ or FITC‐labeled oligonucleotides as mutation probes. Generally between 20—50 nucleotides in length, the probe hybridizes to the target sequence at the reaction temperature. Mismatch repair enzymes (MREs) then cut the probe at the point of mismatch. Once the probe is cleaved, the fragments become thermally unstable and fall off the target, thereby allowing another full‐length probe to hybridize. This oscillating process amplifies the signal (cleaved probe). Cleavage products can be detected by electrophoretic separation followed by autoradiography, or by laser‐induced fluorescence‐capillary electrophoresis (LIF‐CE) of fluorophore‐labeled probes in two minutes using a novel CE matrix. In the present experiments, we employed the mesophilic Escherichia coli enzyme deoxyinosine 3′‐endonuclease (Endo V), and a novel thermostable T/G DNA glycosylase, TDG mismatch repair enzyme (TDG‐MRE). MIDAS differentiated between a clinical sample BRCA1 wild‐type sequence and a BRCA1 185delAG mutation without the need for polymerase chain reaction (PCR). The combination of MIDAS with LIF‐CE should make detection of known point mutations, deletions, and insertions a rapid and cost‐effective technique well suited for automation.

Inhibition of AMV DNA polymerase by polyriboadenylic acid containing ε-adenosine residues

Summary Polyriboadenylic acid was treated with chloroacetaldehyde under conditions known to introduce e-adenosine groups. The degree of modification was monitored by increase in fluorescence intensity. Modified e-poly rA was found to be inhibitory when unprimed 70S AMV RNA was used as a substrate, suggesting direct competition with the poly rA tract of the RNA. Since e-poly rA cannot effectively base pair with nucleic acids normally involved in cellular processes, it has the potential of being useful as an inhibitor of oncogenic viral polymerases.

The complete sequence of the Bacillus amyloliquefaciens proviral H2, BamHI methylase gene.

We have previously isolated a gene from Bacillus amyloliquefaciens which encoded a DNA methylase with specificity for the BantHl site (1). Plasmid and genomic DNA isolated from E. coli HB101 cells harboring a plasmid pBamM2.5 containing this gene was resistant to cleavage by BamWl endonuclease. Expression of this methylase gene is dependent on orientation in pSP64 and under control of the LacZ promoter. The sequence analysis of this gene is shown below. Comparison of this sequence to a partial sequence of a Bacillus amyloliquefaciens prophage encoded methylase indicates that the gene we isolated encodes the prophage H2 BaniHl methylase (2). The sequence contains a 795 base pair open reading frame, nucleotides #441-1236 encoding a 265 amino acid, 30978.7 dalton protein. Deletion analysis of this clone suggests that this is the correct protein. A HindUl-HindlU DNA fragment, nucleotides #1 -651 , was subcloned into pSP64. Clones containing this deletion, which removes 196 carboxyl terminal amino acids, do not express BamHl methylase activity.

Molecular cloning of the intronless EJ ras oncogene using a murine retrovirus shuttle vector

Abstract We have inserted a genomic clone of the human EJ bladder oncogene into a murine retrovirus shuttle vector. Cotransfection of this shuttle vector DNA containing the activated ras oncogene with molecularly cloned Moloney murine leukemia virus into NIH/3T3 cells was able to rescue a replicating and transforming retrovirus. The viral DNA from infected cells was excised by fusion to mouse COP-5 cells and recloned into Escherichia coli as plasmid DNA. Analysis of the recloned plasmids by size, restriction enzyme mapping, and DNA sequence indicated that approximately 5% of the recloned plasmids contained the intronless EJ ras oncogene.

Cloning of the BamHI methyl transferase gene from Bacillus amyloliquefaciens

We wish to report the initial characterization of a recombinant clone containing the BamHI methylase gene. Genomic chromosomal DNA purified from Bacillus amyloliquefaciens was partially cleaved with HindIII, fractionated by size, and cloned into pSP64. Plasmid DNA from this library was challenged with BamHI endonuclease and transformed into Escherichia coli HB101. A recombinant plasmid pBamM6.5 and a subclone pBamM2.5 were shown to contain the BamHI methylase gene based on three independent observations. Both plasmids were found to be resistant to BamHI endonuclease cleavage, and chromosomal DNA isolated from E.coli HB101 cells harboring either of the plasmids pBamM6.5 or pBamM2.5 was resistant to cleavage by BamHI endonuclease. In addition, DNA isolated from lambda phage passaged through E.coli HB101 containing either plasmid was also resistant to BamHI cleavage. Expression of the BamHI methylase gene is dependent on orientation in pSP64. In these clones preliminary evidence indicates that methylase gene expression may be under the direction of the plasmid encoded LacZ promoter.

AVIAN MYELOBLASTOSIS VIRUS (AMV) LINEAR DUPLEX DNA: IN VITRO ENZYMATIC SYNTHESIS AND STRUCTURAL ORGANIZATION ANALYSIS

Publisher Summary This chapter describes the in vitro and enzymatic synthesis and structural organization of avian myeloblastosis virus (AMV) linear duplex DNA. Avian retroviruses contain a unique genome composed of a diploid single-stranded RNA. AMV RNA consists of two 35S subunits which code for at least three viral gene products. These include gag, pol, and env, which code for viral structural proteins, a polymerase, and glycoproteins of the viral envelope, respectively. The identification and location of the leuk gene responsible for leukemogenic transformation remain unknown. In an experiment described in the chapter, full-length cDNA transcripts of intact RNA were isolated after an alkaline sucrose gradient centrifugation. The complete copy is 11% double stranded after nuclease S 1 digestion. A hairpin structure at the 3′ end of the cDNA self-primes synthesis of the second strand. The product of this two-step reaction catalyzed by reverse transcriptase is a linear duplex of 5.2 × 10 6 daltons. The chapter discusses the mechanism of hairpin formation by reverse transcriptase and its possible involvement in proviral synthesis in vivo .