Characterization of the dnmZ, dnmU, and dnmV genes from the daunorubicin-producer Streptomyces peucetius by DNA sequence analysis indicated that these genes encode a protein of unknown function plus a putative thymidine diphospho-4-keto-6-deoxyglucose-3(5)-epimerase and thymidine diphospho-4-ketodeoxyhexulose reductase, respectively. Inactivation of each of the three genes by gene disruption and replacement in the wild-type strain demonstrated that all of them are required for daunosamine biosynthesis.Daunorubicin (DNR) and its C-14-hydroxylated derivative doxorubicin (DXR) (Fig. 1) are clinically important antitumor agents, and like many microbial secondary metabolites, they require a deoxyhexose component for their biological activity (7). These deoxyhexose constituents are commonly 6-deoxyhexoses including 2,6-and 4,6-dideoxy or trideoxy amino hexoses, as is the case for DNR and DXR, which contain the 2,3,6-trideoxy-3-aminohexose daunosamine. The biosynthesis of these biologically important deoxy sugars is not well understood, but progress is being made, fueled in part by the availability of DNA sequence data. These data facilitate the construction of mutant strains that are disrupted in potential sugar genes and permit the assignment of reasonable functions to deduced gene products by sequence comparisons to proteins of known function, such as those for the biosynthesis of rhamnose (16) and the 3,6-dideoxyhexoses (14) that are found in the lipopolysaccharides of gram-negative bacteria. Thus a putative glucose-1-phosphate thymidylyltransferase gene, dnmL (formerly dnrL) (8), which presumably governs the first step of daunosamine biosynthesis (Fig. 1), has been identified in the DNR gene cluster of the wild-type Streptomyces peucetius ATCC 29050 strain, and a thymidine-diphospho (TDP)-glucose-4,6-dehydratase for the formation of the 4-keto-6-deoxyhexulose nucleotide that is a key precursor in deoxyhexose biosynthesis has been purified (28). The gene encoding the putative TDP-glucose-4,6-dehydratase has been localized outside of the DNR gene cluster (8), while a TDP-glucose-4,6-dehydratase homolog, dnmM (formerly dnrM) (8), found adjacent to dnmL, is apparently nonfunctional due to a frameshift mutation. Inactivation of the dnmJ (formerly dnrJ) gene (17) indicates that it is required for daunosamine biosynthesis, and the similarity between DnmJ and AscC (29) suggests that DnmJ is likely to be involved in the addition of the C-3 amino group to a daunosamine precursor (30). Furthermore, disruptions of the dnmT (24) and dnmQ (formerly dnrQ) (21) genes indicate that they too are required for daunosamine biosynthesis although their enzymatic functions have not been established. In this report we extend our investigation of TDP-daunosamine biosynthesis through DNA sequencing and characterization of the dnmZ, dnmU, and dnmV genes that encode a protein of unknown function plus the putative TDP-4-keto-6-deoxyglucose-3(5)-epimerase and TDP-4-ketodeoxyhexulose reductase, respectively, of the daunosamine biosynthetic pathw...

The Streptomyces peucetius dpsY and dnrXgenes govern early and late steps in the biosynthesis of the clinically valuable antitumor drugs daunorubicin (DNR) and doxorubicin (DXR). Although their deduced products resemble those of genes thought to be involved in antibiotic production in several other bacteria, this information could not be used to identify the functions ofdpsY and dnrX. Replacement of dpsYwith a mutant form disrupted by insertion of the aphIIneomycin-kanamycin resistance gene resulted in the accumulation of UWM5, the C-19 ethyl homolog of SEK43, a known shunt product of iterative polyketide synthases involved in the biosynthesis of aromatic polyketides. Hence, DpsY must act along with the other components of the DNR-DXR polyketide synthase to form 12-deoxyaklanonic acid, the earliest known intermediate of the DXR pathway. Mutation ofdnrX in the same way resulted in a threefold increase in DXR production and the disappearance of two acid-sensitive, unknown compounds from culture extracts. These results suggest thatdnrX, analogous to the role of the S. peucetius dnrH gene (C. Scotti and C. R. Hutchinson, J. Bacteriol. 178:7316–7321, 1996), may be involved in the metabolism of DNR and/or DXR to acid-sensitive compounds, possibly related to the baumycins found in many DNR-producing bacteria.

A 2.7 kb BamHl fragment of the daunorubicin biosynthetic cluster in Streptomyces peucetius ATCC 29050 was shown to contain two ORFs, dnrL and dnfM# whose deduced products exhibit a high sequence similarity to a number of glucose-l-phosphate thymidylyl transferases and TDP-D-glucose dehydratases, respectively. Although these genes were believed to be necessary for the synthesis of the deoxyaminosugar, daunosamine, a constituent of daunorubicin, the dnrM gene contains a frameshift in the DNA sequence that causes the premature termination of translation. A gene encoding another TDP-glucose 4#6=dehydratase, previously isolated from 5. peucetius, was identified by PCR amplification of genomic DNA. The presence of this gene explains why a dnrM::aphll mutation did not block daunorubicin production.

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An analysis of critical reading styles of freshmen and senior biology students was compared to that of biology faculty members through the use of highlighting a primary research article. Sentence-by-sentence comparisons were made within each group and the data were analyzed; the composite picture from each group was then compared to the other groups. There appears to be a close agreement of what is deemed important content as judged by faculty but less agreement by seniors and even less agreement by freshmen regarding the value of each line of the text. The results imply that experts in a field appear able to discriminate what is important and valuable in the primary literature and that the novice appears to develop some degree of scientific literacy during his or her undergraduate career.