Joseph Michniewicz

Biosynthesis of ganglioside mimics in Campylobacter jejuni OH4384. Identification of the glycosyltransferase genes, enzymatic synthesis of model compounds, and characterization of nanomole amounts by 600-mhz (1)h and (13)c NMR analysis.

We have applied two strategies for the cloning of four genes responsible for the biosynthesis of the GT1a ganglioside mimic in the lipooligosaccharide (LOS) of a bacterial pathogen,Campylobacter jejuni OH4384, which has been associated with Guillain-Barré syndrome. We first cloned a gene encoding an α-2,3-sialyltransferase (cst-I) using an activity screening strategy. We then used nucleotide sequence information from the recently completed sequence from C. jejuni NCTC 11168 to amplify a region involved in LOS biosynthesis from C. jejuni OH4384. The LOS biosynthesis locus from C. jejuni OH4384 is 11.47 kilobase pairs and encodes 13 partial or complete open reading frames, while the corresponding locus in C. jejuni NCTC 11168 spans 13.49 kilobase pairs and contains 15 open reading frames, indicating a different organization between these two strains. Potential glycosyltransferase genes were cloned individually, expressed in Escherichia coli, and assayed using synthetic fluorescent oligosaccharides as acceptors. We identified genes encoding a β-1,4-N-acetylgalactosaminyl-transferase (cgtA), a β-1,3-galactosyltransferase (cgtB), and a bifunctional sialyltransferase (cst-II), which transfers sialic acid to O-3 of galactose and to O-8 of a sialic acid that is linked α-2,3- to a galactose. The linkage specificity of each identified glycosyltransferase was confirmed by NMR analysis at 600 MHz on nanomole amounts of model compounds synthesized in vitro. Using a gradient inverse broadband nano-NMR probe, sequence information could be obtained by detection of3J(C,H) correlations across the glycosidic bond. The role of cgtA and cst-II in the synthesis of the GT1a mimic in C. jejuni OH4384 were confirmed by comparing their sequence and activity with corresponding homologues in two relatedC. jejuni strains that express shorter ganglioside mimics in their LOS.

Transposition studies of mini-Mu plasmids constructed from the chemically synthesized ends of bacteriophage Mu

We describe below the chemical synthesis of the right and left ends of bacteriophage Mu and characterize the activity of these synthetic ends in mini-Mu transposition. Mini-Mu plasmids were constructed which carry the synthetic Mu ends together with the Mu A and B genes under control of the bacteriophage lambda pL promoter. Derepression of pL leads to a high frequency of mini-Mu transposition (5.6 X 10(-2) which is dependent on the presence of the Mu ends and the Mu A and B proteins. Five deletion mutants in the Mu ends were tested in the mini-Mu transposition system and their effects on transposition are described.

Synthesis of a human insulin gene. VII. Synthesis of preproinsulin-like human DNA, its cloning and expression in M13 bacteriophage.

A 74-bp DNA sequence coding for the pre sequence of human preproinsulin and containing EcoRI termini was synthesized by the chemical enzymatic method, joined with previously synthesized proinsulin DNA, and cloned in the M 13mp8 vector. A clone pNB82 -121 was identified by DNA sequence which confirmed the correct orientation of the pre sequence to the proinsulin DNA. The EcoRI site at the junction of pre- and proinsulin DNA was eliminated by removing a triplet ATT using a synthetic 19-mer primer. To simplify preproinsulin isolation and to study its expression in the M 13 system, a 25-bp affinity leader sequence coding for (glu)7 was inserted at the remaining EcoRI site; this put the preproinsulin DNA in a correct reading frame with the AUG initiation codon of beta-galactosidase. Preproinsulin was expressed under lac promoter control as analyzed by a radioimmunoassay (RIA) against C-peptide.

Separation of synthetic deoxyribo-oligonucleotides by thin layer chromatography

Abstract An easy and rapid method for the purification and identification of complex chemical reaction mixtures of deoxyribo-oligonucleotides has been achieved by using Avicel-Cellulose thin-layer plates.

A novel deletion found during cloning of a synthetic palindromic DNA

A 212-bp palindromic DNA comprising two copies of the left end of bacteriophage Mu was assembled from chemically synthesized oligonucleotides and inserted into plasmid pUC9. When cloned and propagated in Escherichia coli, the palindrome was found to be unstable and was generally lost. However, in a few cases, a precise, asymmetric deletion of one half of the insert was observed. This pattern of deletion suggests that the symmetry axis region of the palindrome was involved as recognition site in the deletion process.

Retention of enzymatic activity by N-terminal domain (1–78) T4-lysozyme: Expression of synthetic DNA in Escherichiacoli

DNA of 235 b.p. coding for N-terminal domain (1-78) T4-lysozyme was synthesized and cloned by ligating twelve synthetic fragments with a linearized plasmid pUCE8 followed by transformation. On expression in E. coli strain JM103 cells, colonies containing the synthetic DNA were found to be lytic. On purification, clone ptly. 23-5 was found to contain polypeptide (M.W. 10,500), corresponding to N-terminal domain, its dimeric and aggregate form. It was identified by amino acid sequence analysis of the dimeric form.

“In vivtro” method of assembling a synthetic gene

Without prior in vitro enzymatic ligation a DNA duplex was assembled successfully by directly transforming competent cells with a mixture containing six synthetic complimentary oligodeoxyribonucleotides and a linearized plasmid. One out of 100 transformants was positive in colony hybridization with one of the synthetic fragment probe. The sequence of the DNA duplex inserted into the plasmid was confirmed by dideoxy sequencing method.

Use of fluorene-9-methanol as a phosphate protecting group in the synthesis of deoxyribo-oligonucleotides

The fluore-9-methanol esters of the 5′-phosphates of oligonucleotides are soluble in organic solvents, and the protected oligonucleotides can be isolated by solvent extraction from the reaction mixture.