Crystal structure of the obese protein leptin-E100.

Mutations in the obese gene (OB) or in the gene encoding the OB receptor(OB-R) result in obesity, infertility and diabetes in a variety of mouse phenotypes. The demonstration that OB protein (also known as leptin) can normalize body weight in ob/ob mice has generated enormous interest. Most human obesity does not appear to result from a mutant form of leptin: rather, serum leptin concentrations are increased and there is an apparent inability to transport it to the central nervous system (CNS). Injection of leptin into the CNS of overfed rodents resistant to peripheral administration was found to induce biological activity. Consequently, for the leptin to act as a weight-lowering hormone in human obesity, it appears that appropriate concentrations must be present in the CNS. This places a premium on understanding the structure of the hormone in order to design more potent and selective agonists. Here we report the crystal structure at 2.4A resolution of a human mutant OB protein (leptin-E100) that has comparable biological activity to wild type but which crystallizes more readily. The structure reveals a four-helix bundle similar to that of the long-chain helical cytokine family.

Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp.

We have constructed cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. All vectors contain the 760-bp oriT fragment from the IncP plasmid, RK2. Transfer functions need to be supplied in trans by the E. coli donor strain. We have incorporated into these vectors selectable antibiotic-resistance markers (AmR, ThR, SpR) that function in Streptomyces spp. and other features that should allow for: (i) integration via homologous recombination between cloned DNA and the Streptomyces spp. chromosome, (ii) autonomous replication, or (iii) site-specific integration at the bacteriophage phi C31 attachment site. Shuttle cosmids for constructing genomic libraries and bacteriophage P1 cloning vector capable of accepting approx. 100-kb fragments are also described. A simple mating procedure has been developed for the conjugal transfer of these vectors from E. coli to Streptomyces spp. that involves plating of the donor strain and either germinated spores or mycelial fragments of the recipient strain. We have shown that several of these vectors can be introduced into Streptomyces fradiae, a strain that is notoriously difficult to transform by PEG-mediated protoplast transformation.

Production of a hybrid macrolide antibiotic in Streptomyces ambofaciens and Streptomyces lividans by introduction of a cloned carbomycin biosynthetic gene from Streptomyces thermotolerans.

The structurally related macrolide antibiotics carbomycin (Cb) and spiramycin (Sp) are produced by Streptomyces thermotolerans and Streptomyces ambofaciens, respectively. Both antibiotics contain 16-membered lactone rings to which deoxysugars are attached. There are three sugars in Sp (forosamine, mycaminose and mycarose) and two sugars in Cb (mycaminose and a derivative of mycarose containing an isovaleryl group at position 4). We have identified the gene from S. thermotolerans (designated carE), which appears to encode an enzyme that acylates this mycarose sugar, and have shown that recombinant strains containing carE can use Sp as a substrate and convert it to the hybrid antibiotic, isovaleryl Sp (ivSp). Expression of carE was demonstrated in two heterologous hosts: in S. ambofaciens, where endogenously synthesized Sp was converted to ivSp, and in Streptomyces lividans where exogenously added Sp was converted to ivSp. The carE gene was isolated on a cosmid that also encodes genes required for Cb-lactone formation. These genes reside on a DNA segment of about 70 kb and are part of a Cb biosynthetic gene cluster that is flanked by two Cb-resistance genes, carA and carB. Mapping studies and nucleotide sequence analysis revealed that carE is located at one end of this gene cluster, immediately adjacent to the carB gene. Genes carB and carE are transcribed convergently and may share a common transcriptional terminator sequence.

Cloning and nucleotide sequence of a carbomycin-resistance gene from Streptomyces thermotolerans.

Two plasmids (pOJ158 and pOJ159) containing DNA fragments from the carbomycin(Cb)-producing strain Streptomyces thermotolerans were identified in Streptomyces griseofuscus based on their ability to confer resistance to Cb. The Cb-resistance determinants on pOJ158 and pOJ159 were designated carA and carB, respectively. In S. griseofuscus, pOJ159 also confers resistance to spiramycin, rosaramicin, lincomycin, and vernamycin B, but not to tylosin; in Streptomyces lividans, pOJ159 additionally confers resistance to erythromycin and oleandomycin. The carB gene was localized on pOJ159 to a 1.25-kb region whose nucleotide sequence was determined. The sequence has a G + C content of 68% and contains the coding sequence for carB and portions of the 5' and 3' untranslated regions. A comparison of the amino acid sequence of the protein encoded by carB (as deduced from the nucleotide sequence) with the deduced amino acid sequence of the RNA methylase from Streptomyces erythraeus (encoded by ermE) revealed extensive homology, suggesting that carB also encodes an RNA methylase. The region 5' to the coding sequence does not contain a small ORF or regions of complementarity that are commonly associated with translationally regulated macrolide-lincosamide-streptogramin B resistance genes. The 3' untranslated region contains an inverted repeat sequence that potentially can form a stable RNA stem-loop structure with a calculated delta G of -70 kcal.

Translation of a synthetic two-cistron mRNA in Escherichia coli.

A synthetic two-cistron expression system was constructed for the high-level expression of eukaryotic genes in Escherichia coli. This system was designed to overcome translational inhibition of mRNAs containing eukaryotic sequences. The first cistron in this system is a 31-base A + T-rich synthetic sequence that provides for efficient translation initiation. The second cistron contains the protein coding sequence for the eukaryotic gene. Insertion of the first cistron between the 5' untranslated region of the mRNA and the protein coding region separates the two and thereby potentially minimizes the formation of local secondary structures that might prevent ribosomes from binding and initiating translation. The 31-base cistron contains three nonsense codons (TAA), one in each of the three translational reading frames, and an 8-base Shine-Dalgarno sequence that is complementary to the 3' end of the 16S rRNA. The effects of translation of the first cistron in all three reading frames on the expression of the second cistron was examined. The most efficient expression of the second cistron seemed to occur when the stop codon that terminates translation of the first cistron is located 3' to the Shine-Dalgarno sequence and close to the AUG start codon for the second cistron. When the Shine-Dalgarno sequence was deleted from the first cistron, no detectable expression of the second cistron was observed. This two-cistron system has been used to express the gene encoding methionylalanyl bovine growth hormone with its native codons and the gene encoding methionyl human growth hormone at a level greater than 20% of total cell protein. In the case of human growth hormone, we show that the amount of gene product is not significantly diminished by placing a "functional" first cistron in front of a gene that can be expressed without a cistron.

Role of mRNA translational efficiency in bovine growth hormone expression in Escherichia coli.

The conditions necessary for high-level expression of methionyl bovine growth hormone (Met-bGH) in Escherichia coli were investigated. Plasmids were constructed that contain a thermoinducible runaway replicon and either the E. coli tryptophan or lipoprotein promoter and ribosome binding sites, which served as transcriptional and translational initiation sites for the expression of the bGH gene. The expression of Met-bGH was low with either system. However, expression levels of up to 30% of total cell protein were obtained after the introduction of additional codons 3' to the initiating AUG codon, thus altering the NH2-terminal amino acid sequence of bGH. To obtain high-level expression of Met-bGH a two-cistron system was constructed in which the codons that enhanced the expression of bGH were incorporated into the first cistron, and the coding region for Met-bGH was incorporated into the second cistron. This approach may be generally applicable to achieving high-level expression of a gene that contains NH2-terminal sequences that do not allow for its efficient expression. Analyses of the stabilities of the bGH derivatives and their transcripts in vivo suggested that the variations in the level of expression were due to variations in the efficiency of mRNA translation.