No abstract

The pathogenic yeast, Candida albicans, is insensitive to the anti-mitotic drug, benomyl, and to the dihydrofolate reductase inhibitor, methotrexate. Genes responsible for the intrinsic drug resistance were sought by transforming Saccharomyces cerevisiae, a yeast sensitive to both drugs, with genomic C. albicans libraries and screening on benomyl or methotrexate. Restriction analysis of plasmids isolated from benomyl- and methotrexate-resistant colonies indicated that both phenotypes were encoded by the same DNA fragment. Sequence analysis showed that the fragments were nearly identical and contained a long open reading frame of 1694 bp (ORF1) and a small ORF of 446 bp (ORF2) within ORF1 on the opposite strand. By site-directed mutagenesis, it was shown that ORF1 encoded both phenotypes. The protein had no sequence similarity to any known proteins, including beta-tubulin, dihydrofolate reductase, and the P-glycoprotein of the multi-drug resistance family. The resistance gene was detected in several C. albicans strains and in C. stellatoidea by DNA hybridization and by the polymerase chain reaction.

The complete nucleotide sequence of the type I dihydrofolate reductase gene from Tn7 was determined. The structural gene coded for a polypeptide of 157 amino acid residues. The polypeptide deduced from the DNA sequence had a molecular weight of 17,577 which was in good agreement with that estimated by mobility in SDSpolyacrylamide gels. Sequences were identified proximal to the coding region which were similar to those found in the consensus E. coli promoter region and for the initiation of protein synthesis. Features consistent with the termination of RNA transcription were present distal to the structural gene. No homology was apparent when the DNA sequence of the type I gene was compared to the sequence of the type II plasmid DHFR genes, but sequence homology was evident when the type I and E. coli chromosomal enzymes were compared. Homology was greatest in the regions coding for amino acids which in the E. coli chromosomal enzyme are associated with substrate, cofactor and inhibitor binding.

Resistance of Escherichia coli to trimethoprim (TMP)-sulfamethoxazole remains at 3%-8% at many medical centers within the United States. In this study a 44% resistance rate was observed among E. coli isolated at a pediatric hospital in Santiago, Chile, and a 40% resistance rate at a general teaching hospital in Bangkok, Thailand. Most isolates were from urinary tract infections and showed high-level resistance (minimal inhibitory concentration of TMP greater than 1,000 micrograms/ml). Nineteen of 35 isolates tested transferred resistance to TMP; most cotransferred resistance to streptomycin and sulfonamides. Dihydrofolate reductase type I was detected by gene probing in 14 of 35 strains. Subsequent investigations in Brazil, Honduras, and Costa Rica revealed that this high rate of resistance was not an isolated phenomenon.

The nucleotide sequence of a transposon Tn7 DNA fragment encoding a 3"(9)-O-nucleotidyltransferase, an aminoglycoside-modifying enzyme, which mediates bacterial resistance to spectinomycin and streptomycin, was determined. The aadA structural gene was 786 bases long and predicted a polypeptide of 262 amino acids with a calculated molecular weight of 29,207. Comparison of the DNA sequences of Tn7 and plasmid R538-1 indicated that their aadA genes were nearly identical. Comparison of the polypeptides predicted by the aadA genes of Tn7 and Tn554 indicated that the genes were related.

Trimethoprim (TMP) resistance among Shigella species isolated from Finnish travelers increased from 3.0% in 1975-1982 to 42.0%-43.8% in 1987-1988. Of the 317 TMP-resistant Shigella isolates identified during 1975-1988, 175 (55%) collected in 1985-1987 and in 1988 were tested further. Almost all (98%) were highly resistant to TMP, suggesting a plasmid-mediated origin. The type I dihydrofolate reductase (DHFR) gene was detected in 85% of the isolates studied. Twenty-three percent of the type I DHFR-positive isolates failed to hybridize with a probe detecting only Tn7-derived sequences, suggesting that the type I DHFR gene may occur independently of transposon Tn7. Four of the five Shigella species isolated from travelers to Sri Lanka hybridized with the probe for type V DHFR gene, implying a local distribution of the type V DHFR gene. The type II and type III DHFR genes were not found among the isolates studied. Only 12% of the TMP-resistant Shigella isolates failed to hybridize with any of the DHFR gene probes used.

We have engineered a cysteine residue at position 442 (EU/OU numbering) in the third constant domain (C H 3) of the heavy chain of several IgGs with different specificities, isoforms, and variants with the intent to introduce a site for chemical conjugation. The variants were expressed in NS0 mouse myeloma cells, where monomeric IgG is the major form and formation of aggregate was minimal. Monomeric IgG contained no free thiol; however, it was discovered that the engineered thiols were reversibly blocked and could be reduced under controlled conditions. Following reduction, reactive thiol was conjugated with a cysteine-specific bifunctional chelator, bromoacetyl-TMT to a humanized 323/A3 IgG4 variant. Conjugation had no significant effect on antibody affinity. To prove that the conjugation was site-specific, an antibody-TMT conjugate was labeled with lutetium-177 and subjected to peptide mapping followed by sequence analysis. Glu-C digestion demonstrated that 91% of the label was recovered in the COOH-terminal peptide fragment containing the engineered cysteine.

The ability to specifically target a cell-type is important for the development of vectors for in vivo gene therapy. In order to produce retrovirus vectors targeting ovarian cancer cells, which specifically overexpress ␣ folate receptor (␣FR), a single chain antibody was fused as an N-terminal extension of the ecotropic and amphotropic murine leukemia virus (MLV) envelope glycoproteins. Vector particles bearing the modified glycoproteins were produced and analysed. Although conventional FACS studies indicated that viral particles bearing the modified Env could bind to ovarian cancer cells, targeted infection was not achieved. The initial step of viruscell interaction was further studied using an immunofluorescence technique, which allows visualisation of single retrovirus particles. Vectors bearing chimeric or wild-type