A 5.9-kb tandem repeat at the euchromatin-heterochromatin boundary of the X chromosome of Drosophila melanogaster.

We present an analysis of a chromosomal walk in the region of the euchromatin-heterochromatin transition at the base of the X chromosome of Drosophila melanogaster. This region is difficult to analyse because of the presence of repeated sequences, and we have used cosmids to walk from the last euchromatic gene, suppressor of forked, towards the pericentric heterochromatin. The proximal 30-kb sequence we have isolated consists of repetitive DNA, including four tandem copies of a 5.9-kb sequence. This tandem repeat is itself a mosaic of other, mostly repeated, sequences, including part of a retrotransposon without long terminal repeats, a simple-sequence region of TAA repeats and part of a retrotransposon with long terminal repeats that has not been previously described. Although sequences homologous to these components are found elsewhere in the genome, this arrangement of repeated sequences is only found at the base of the X chromosome. It is conserved in D. melanogaster strains of different geographic origin, but is not conserved in even closely related species.

Gene flow on the ice: genetic differentiation among Adélie penguin colonies around Antarctica.

Each summer Adélie penguins breed in large disjunct colonies on ice-free areas around the Antarctic continent. Comprising > 10 million birds, this species represents a dominant feature of the Antarctic ecosystem. The patchy distribution within a large geographical range, natal philopatry and a probable history of refugia, suggest that this species is likely to exhibit significant genetic differentiation within and among colonies. We present data from seven microsatellite DNA loci for 442 individuals from 13 locations around the Antarctic continent. With the exception of one locus, there was no significant genic or genotypic heterogeneity across populations. Pairwise FST values were low with no value > 0.02. When all colonies were compared in a single analysis, the overall FST value was 0.0007. Moreover, assignment tests were relatively ineffective at correctly placing individuals into their respective collection sites. These data reveal a lack of genetic differentiation between Adélie penguin colonies around the Antarctic continent, despite substantial levels of genetic variation. We consider this homogeneity in terms of the dispersal of individuals among colonies and the size of breeding groups and discuss our results in terms of the glacial history of Antarctica.

The design and synthesis of thrombin inhibitors: analogues of MD805 containing non-polar surrogates for arginine at the P1 position.

A series of monocyclic and bicyclic amino acids have been synthesised and incorporated into thrombin inhibitors based on CGH728, an analogue of the Mitsubishi compound MD805. Benzthiazolylalanine (Bta) was found to be a good non-polar substitute for arginine at the P1 position, yielding compounds with low nanomolar potency and good selectivity for thrombin.

The design and synthesis of thrombin inhibitors: the introduction of in vivo efficacy and oral bioavailability into benzthiazolylalanine inhibitors.

The further optimisation of the novel lead compound CGH752 (Fig. 1) is described. By introducing various substituents into the 6-position of the 3,3-dimethyltetrahydroquinoline (DMTHQS) ring we have been able to favourably affect the in vitro and in vivo activity, and the pharmacokinetics of such compounds. One of the inhibitors synthesised (CGH1484) is bioavailable and shows efficacy in animal models of thrombosis.

Repair of elastase-digested elastic fibers in acellular matrices by replating with neonatal rat-lung lipid interstitial fibroblasts or other elastogenic cell types.

Disruption of elastic fibers is a major factor in the pathogenesis of pulmonary emphysema. Elastic fibers in culture, injured by exposure to elastase, undergo repair in the presence of elastogenic cells that restores the fibers toward normal as determined by biochemical and ultrastructural methods. The repair appears to be the result of both salvage and de novo repair mechanisms. The evidence for salvage repair is that hot-alkali resistance, lost as a result of elastase treatment, is restored to previously radiolabeled elastic fibers. This repair mechanism has been shown in aortic smooth muscle cell cultures. In order to determine the potential relevance of this mechanism for elastic fiber repair in the lungs, experiments were carried out using neonatal rat lung lipid interstitial fibroblasts (LIF). Treatment of the LIF cultures with elastase, in the absence of serum, caused solubilization of 12% of elastin; however, 81% of the elastin protein and 80% of the elastin-associated radioactivity (EAR) were solubilized by subsequent hot-alkali treatment, indicating that most of the elastin was retained in the matrix but was damaged. Ultrastructurally, the elastic fibers were frayed. After 6 additional wk in culture, hot-alkali resistant elastin protein and EAR were restored to 88 and 62% of control values, respectively, and the ultrastructural appearance of elastic fibers was restored to normal. We calculate that about 42% of the restored elastin represented salvage repair; the remainder was new elastin. No repair occurred in matrices rendered acellular by azide treatment; however, when acellular matrices were replated with LIF, repair was complete at 6 wk. No repair was seen when acellular matrices were replated with a transformed mouse macrophage cell line. We conclude that lung LIF are capable of mounting a robust repair process after elastolytic injury of elastin and that the repair is the result of both salvage and de novo repair mechanisms.

A 1.5 kb repeat sequence flanks the suppressor of forked gene at the euchromatin-heterochromatin boundary of the Drosophila melanogaster X chromosome.

A 1.5 kilobasepair repeated DNA sequence is duplicated in direct orientation so as to flank the suppressor of forked gene in the euchromatin-heterochromatin transition region on the X chromosome of Drosophila melanogaster. These two copies are almost identical, but DNA blotting, analysis of cloned sequences and database searches show that elsewhere in the genome, homologous sequences are poorly conserved. They are often associated with other repeats, suggesting that they may belong to a scrambled and clustered middle repetitive DNA family. The sequences do not appear to be related to transposable elements and their location in different strains is conserved. In situ hybridization to metaphase chromosomes shows that homologous sequences are concentrated in the pericentric regions of the autosomes and the X chromosome. The sequences are not significantly under-represented in DNA from polytene tissue and must lie in the replicated regions of polytene chromosomes. The almost perfect conservation of the two repeats around suppressor of forked in D. melanogaster suggests they arose by duplication or gene conversion. Suppression of recombination in this chromosomal region presumably allows this unusual organization to be stably maintained. In the X-ray induced allele, suppressor of forked-L26, the sequence between the repeats, including the gene, and one copy of the repeat have been deleted.

Interallelic complementation at the suppressor of forked locus of Drosophila reveals complementation between suppressor of forked proteins mutated in different regions.

The Su(f) protein of Drosophila melanogaster shares extensive homologies with proteins from yeast (RNA14) and man (77 kD subunit of cleavage stimulation factor) that are required for 3' end processing of mRNA. These homologies suggest that su(f) is involved in mRNA 3' end formation and that some aspects of this process are conserved throughout eukaryotes. We have investigated the genetic and molecular complexity of the su(f) locus. The su(f) gene is transcribed to produce three RNAs and could encode two proteins. Using constructs that contain different parts of the locus, we show that only the larger predicted gene product of 84 kD is required for the wild-type function of su(f). Some lethal alleles of su(f) complement to produce viable combinations. The structures of complementing and noncomplementing su(f) alleles indicate that 84-kD Su(f) proteins mutated in different domains can act in combination for partial su(f) function. Our results suggest protein-protein interaction between or within wild-type Su(f) molecules.

Homology with Saccharomyces cerevisiae RNA14 suggests that phenotypic suppression in Drosophila melanogaster by suppressor of forked occurs at the level of RNA stability.

The suppressor of forked [su(f)] locus of Drosophila melanogaster encodes at least one cell-autonomous vital function. Mutations at su(f) can affect the expression of unlinked genes where retroviral-like transposable elements are inserted. Changes in phenotype are correlated with changes in mRNA profiles, indicating that su(f) affects the production and/or stability of mRNAs. We have cloned the su(f) gene by P-element transposon tagging. Alterations in the DNA map of eight lethal alleles were detected in a 4.3-kb region. P-element-mediated transformation using a fragment including this interval rescued all aspects of the su(f) mutant phenotype. The gene is transcribed to produce a major 2.6-kb RNA and minor RNAs of 1.3 and 2.9 kb, which are present throughout development, being most abundant in embryos, pupae, and adult females. The major predicted gene product is an 84- kD protein that is homologous to RNA14 of Saccharomyces cerevisiae, a vital gene where mutation affects mRNA stability. This suggests that phenotypic modification by su(f) occurs at the level of RNA stability.

Molecular and cytogenetic analysis of the heterochromatin-euchromatin junction region of the Drosophila melanogaster X chromosome using cloned DNA sequences.

We have used three cloned DNA sequences consisting of (1) part of the suppressor of forked transcription unit, (2) a cloned 359-bp satellite, and (3), a type I ribosomal insertion, to examine the structure of the base of the X chromosome of Drosophila melanogaster where different chromatin types are found in juxtaposition. A DNA probe from the suppressor of forked locus hybridizes exclusively to the very proximal polytenized part of division 20, which forms part of the beta-heterochromatin of the chromocenter. The cloned 359-bp satellite sequence, which derives from the proximal mitotic heterochromatin between the centromere and the ribosomal genes, hybridizes to the under replicated alpha-heterochromatin of the chromocenter. The type I insertion sequence, which has major locations in the ribosomal genes and in the distal mitotic heterochromatin of the X chromosome, hybridizes as expected to the nucleolus but does not hybridize to the beta-heterochromatic division 20 of the polytene X chromosome. Our molecular data reveal that the suppressor of forked locus, which on cytogenetic grounds is the most proximal ordinary gene on the X chromosome, is very close to the junction of the polytenized and non-polytenized region of the X chromosome. The data have implications for the structure of beta-heterochromatin-alpha-heterochromatin junction zones in both mitotic and polytene chromosomes, and are discussed with reference to models of chromosome structure.

Structural analysis of Doc transposable elements associated with mutations at the white and suppressor of forked loci of Drosophila melanogaster.

DNA sequences from two spontaneous mutations of Drosophila melanogaster associated with insertion of a Doc transposable element have been cloned. In white-one, the element is inserted in the white locus close to where transcription initiates. In a lethal allele of suppressor of forked, su(f)S2, the element is inserted within the transcription unit in the protein coding region. Four other Doc elements have been cloned from a wild-type strain. Doc is a member of the class of transposable elements known as retroposons, which includes the D. melanogaster F, G, Jockey, and I elements. There is no sequence homology between the ends of the Doc element. The 3' or right end terminates with a polyadenylation signal sequence followed by a stretch of oligo-A. The length of the oligo-A varies between elements, and a duplication of variable size is found as a direct repeat flanking inserted Doc elements. Members of the family are conserved at the 3' end, but may be truncated at the 5' or left end. These structural features suggest a mechanism of transposition via an RNA intermediate.

Activities of some key enzymes of carbohydrate, ketone body, adenosine and glutamine metabolism in liver, and brown and white adipose tissues of the rat.

In general, the activities of enzymes in brown adipose tissue (BAT) are more similar to those in white adipose tissue than those in liver. Thus the activities of the glycolytic enzymes hexokinase and 6-phosphofructokinase are high but those of glucose 6-phosphatase and fructose bisphosphatase are non-detectable in the two adipose tissues. The activity of HMG-CoA synthase was non-detectable in BAT indicating that this tissue, unlike liver, cannot produce ketone bodies from fatty acid oxidation but, since the tissue possesses a high activity of HMG-CoA lyase, it might produce ketone bodies from leucine catabolism. The findings suggest that 'metabolically' brown adipose tissue can be classified better as an adipose tissue than as a peripheral liver. A high activity of 3-oxoacid CoA transferase but a non-detectable activity of 3-hydroxybutyrate dehydrogenase suggests that BAT can utilise acetoacetate but not 3-hydroxybutyrate for heat generation during cold exposure plus starvation.