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The doublesex (dsx) gene of Drosophila melanogaster encodes both male‐specific and female‐specific polypeptides, whose synthesis is regulated by alternative sex‐specific splicing of the primary dsx transcript. The alternative splicing of the dsx mRNA is the last known step in a cascade of regulatory gene interactions that involves both transcriptional and post‐transcriptional mechanisms. Genetic studies have shown that the products of the dsx locus are required for correct somatic sexual differentiation of both sexes, and have suggested that each dsx product functions by repressing expression of terminal differentiation genes specific to the opposite sex. However, these studies have not shown whether the dsx gene products function directly to regulate the expression of target genes, or indirectly through another regulatory gene. We report here that the male‐ and female‐specific DSX proteins, expressed in E.coli, bind directly and specifically in vitro to three DNA sequences located in an enhancer region that regulates female‐specific expression of two target genes, the yolk protein genes 1 and 2. This result suggests strongly that dsx is a final regulatory gene in the hierarchy of regulatory genes controlling somatic sexual differentiation.

A permanent, ordered collection of 23,000 recombinant DNA plasmids containing Drosophila melanogaster DNA has been established. Simple and practical methods for storing and manipulating this collection were developed. In addition, an improved, simple and inexpensive method for making paper filter replicas of such an ordered collection and of a high density (10,000 colonies/petri dish) unordered collection was developed. These filter replicas are suitable for nucleic acid hybridization screens of recombinant DNA colinies and each filter replica can be used for many (greater than 5) successive screens. The kinetics of this hybridization reaction were examined and allow design of experiments that detect colony complementarity to a nucleic acid that is 0.5% of the hybridization probe.

Two enzymes, designated, E-2 and E-2, catalyze different oxidation reactions of an aci-reductone intermediate in the methionine salvage pathway. E-2 and E-2, overproduced in Escherichia coli from the same gene, have the same protein component. E-2 and E-2 are separable on an anion exchange column or a hydrophobic column. Their distinct catalytic and chromatographic properties result from binding different metals. The apo-enzyme, obtained after metal is removed from either enzyme, is catalytically inactive. Addition of Ni 2؉ or Co 2؉ to the apo-protein yields E-2 activity. E-2 activity is obtained when Fe 2؉ is added. Production in intact E. coli of E-2 and E-2 depends on the availability of the corresponding metals. These observations suggest that the metal component dictates reaction specificity.S-Methylthioadenosine, a metabolite derived from methionine (1, 2), is a strong inhibitor of polyamine biosynthesis and transmethylation reactions (2, 3). Therefore its concentration in biological systems must be tightly controlled. Control is achieved through a ubiquitous metabolic pathway called the methionine salvage pathway, which catalyzes conversion of the 5-methylthio-D-ribose moiety of S-methylthioadenosine to methionine (4 -7).In Klebsiela pneumoniae where all intermediates of the pathway have been identified (4 -7), metabolism of an aci-reductone is a branch point in the pathway ( Fig. 1) (8 -10). This molecule can undergo either a 1,2-oxygenlytic reaction to yield the ␣-keto acid precursor of methionine (Reaction 1) or a 1,3-oxygenlytic reaction to yield CO, formate, and methylthiopropionic acid (Reaction 2). The purpose of the off-pathway transformation of the aci-reductone (Reaction 2) is unclear. CO may simply be an easily cleared byproduct. Recent experiments in mammals, however, have established CO as a diffusible neurotransmitter, acting in a similar manner to that of nitric oxide (11,12). CO may also play a role as a messenger in bacteria.In a previous study we purified E-2, which catalyzes Reaction 2, to near homogeneity from K. pneumoniae (8). To study the structure and catalytic action of E-2, we decided to clone and overproduce the enzyme in Escherichia coli. To our surprise, E-2Ј which catalyzes Reaction 1, the other branch of the pathway, is overproduced in the same E. coli cells. In this report we describe the purification and characterization of both enzymes and demonstrate that their distinct catalytic and chromatographic properties result from binding different metals. EXPERIMENTAL PROCEDURESExpression and Purification of E-2 and E-2Ј-The E-2 gene was inserted at the initiator methionine codon of pET11a (13), a T7 RNA polymerase expression system. E. coli strain BL21, transformed with pET11a-E2, was grown in M9 medium (14) containing 100 mg/liter ampicillin at 37°C. When the absorbance at 600 nm reached 0.35, 0.3 mM isopropyl-D-thiogalactoside was added to induce E-2 expression at 25°C. The cells were incubated for an additional 12 h at 25°C, harvested by centrifugation, and stored frozen a...

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