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L M Blatt

Publications and source records attributed to L M Blatt.

49 records · Page 3Linked to original sources
PubMed1991

Cloning and characterization of a Pseudomonas mendocina KR1 gene cluster encoding toluene-4-monooxygenase.

Pseudomonas mendocina KR1 metabolizes toluene as a carbon source by a previously unknown pathway. The initial step of the pathway is hydroxylation of toluene to form p-cresol by a multicomponent toluene-4-monooxygenase (T4MO) system. The T4MO enzyme system has broad substrate specificity and provides a new opportunity for biodegradation of toxic compounds and bioconversions. Its known activities include conversion of a variety of phenyl compounds into the phenolic derivatives and the complete degradation of trichloroethylene. We have cloned and characterized a gene cluster from KR1 that determines the offO activity. To clone the T4MO genes, KR1 DNA libraries were constructed in Escherichia coli HB101 by using a broad-host-range vector and transferred to a KR1 mutant able to grow on p-cresol but not on toluene. An insert consisting of two SacI fragments of identical size (10.2 kb) was shown to complement the mutant for growth on toluene. One of the SacI fragments, when cloned into the E. coli vector pUC19, was found to direct the synthesis of indigo dye. The indigo-forming property was correlated with the presence of T4MO activity. The T4MO genes were mapped to a 3.6-kb region, and the direction of transcription was determined. DNA sequencing and N-terminal amino acid determination identified a five-gene cluster, tmoABCDE, within this region. Expression of this cluster carrying a single mutation in each gene demonstrated that each of the five genes is essential for T4MO activity. Other evidence presented indicated that none of the tmo genes was involved in the regulation of the tmo gene cluster, in the control of substrate transport for the T4MO system, or in major processing of the products of the tmo genes. It was tentatively concluded that the tmoABCDE genes encode structural polypeptides of the T4MO enzyme system. One of the tmo genes was tentatively identified as a ferredoxin gene.

Amino Acid Sequence↗
PubMedDate not supplied

Gene therapy with an adeno-associated virus carrying an interferon gene results in tumor growth suppression and regression.

Adeno-associated virus (AAV) vectors were constructed containing both a synthetic type I interferon gene, (IFN-con1) and the bacterial neomycin-resistant gene. Recombinant virions were used to infect a number of human tumor cell lines, including 293, Hela, K562, and Eskol (a hairy cell leukemia-like cell), and geneticin-resistant cells were selected. All IFN-con1-transduced cell lines produced low levels of IFN-con1 and grew at the same rate as nontransduced cell lines. Although these cell lines were resistant to IFN in vitro, when injected into nude mice, 293, K562, and Eskol cells failed to form tumors up to 3 months after the initial inoculum, although mice receiving nontransduced cells developed tumors within 7 to 10 days. Transduced Hela cells grew much slower in vivo and formed much smaller tumors than did the parental cells. When equal numbers of transduced and nontransduced cells were injected into nude mice, tumors initially developed slowly and then completely regressed. Treatment of an established Eskol tumor (histologically a malignant immunoblastic lymphoma) with AAV/IFN-con1-transduced 293 cells resulted in tumor regression, whereas treatment of Eskol tumors with IFN-con1 resulted in a small decrease in tumor size. These results indicate that the human IFN-con1 gene in a viral vector can be used successfully in the treatment of tumors both directly and by tumor-targeted gene therapy.

Animals↗