The specific organisation of satellite DNA sequences on the X-chromosome of Mus musculus: partial independence of chromosome evolution

DNA was isolated from a chinese hamster/mouse hybrid cell line containing a single mouse chromosome, the X-chromosome, and digested with a variety of restriction endonucleases known to cut mouse satellite DNA. After agarose gel electrophoresis and transfer to nitrocellulose, hybridisation was carried out to a radioactive mouse satellite DNA probe. In this manner the organisation of satellite sequences at an individual chromosome was determined. We have found that the organisation of centromeric satellite DNA sequences on the mouse X-chromosome differs from that of other chromosomes in the complement. The nature of the differences suggests features of evolution of highly repeated sequences within a karyotype.     

A PATERNITY TEST CASE FOR THE KILLER WHALE (ORCINUS ORCA) BY DNA FINGERPRINTING

The minisatellite DNA profiles from four captive killer whales (two adult males, a female and her calf) were compared to determine paternity between two potential fathers. One of the males was clearly excluded, while the other shared all paternal bands with the calf. The background of this technique, and its potential applications in captive breeding programs and field studies are discussed.     

Complete sequences of the rRNA genes of Drosophila melanogaster

In this, the first of three papers, we present the sequence of the ribosomal RNA (rRNA) genes of Drosophila melanogaster. The gene regions of D. melanogaster rDNA encode four individual rRNAs: 18S (1,995 nt), 5.8S (123 nt), 2S (30 nt), and 28S (3,945 nt). The ribosomal DNA (rDNA) repeat of D. melanogaster is AT rich (65.9% overall), with the spacers being particularly AT rich. Analysis of DNA simplicity reveals that, in contrast to the intergenic spacer (IGS) and the external transcribed spacer (ETS), most of the rRNA gene regions have been refractory to the action of slippage-like events, with the exception of the 28S rRNA gene expansion segments. It would seem that the 28S rRNA can accommodate the products of slippage-like events without loss of activity. In the following two papers we analyze the effects of sequence divergence on the evolution of (1) the 28S gene "expansion segments" and (2) the 28S and 18S rRNA secondary structures among eukaryotic species, respectively. Our detailed analyses reveal, in addition to unequal crossing-over, (1) the involvement of slippage and biased mutation in the evolution of the rDNA multigene family and (2) the molecular coevolution of both expansion segments and the nucleotides involved with compensatory changes required to maintain secondary structures of RNA.      

Evolution of the cetacean mitochondrial D-loop region.

We sequenced the mitochondrial DNA D-loop regions from two cetacean species and compared these with the published D-loop sequences of several other mammalian species, including one other cetacean. Nucleotide substitution rates, DNA sequence simplicity, possible open reading frames (ORFs), and potential RNA secondary structure were investigated. The substitution rate is an order of magnitude lower than would be expected on the basis of reports on human sequence variation in this region but are consistent with interspecific primate and rodent D-loop sequence variation and with estimates of substitution rates from whole mitochondrial genomes. Deletions/insertions are less common in the cetacean D-loop than in other vertebrate species. Areas of high sequence simplicity (clusters of short repetitive motifs) across the region correspond to areas of high sequence divergence. Three regions predicted to form secondary structures are homologous to such putative structures in other species; however, the presumptive structures most conserved in cetaceans are different from those reported for other taxa. While all three species have possible long ORFs, only a short sequence of seven amino acids is shared with other mammalian species, and those changes that had occurred within it are all nonsynonymous. We conclude that DNA slippage, in addition to point mutation, contributes to the evolution of the D-loop and that regions of conserved secondary structure in cetaceans and an ORF are unlikely to contribute significantly to the conservation of the central region.     

X-ray microtomography of biological tissues using laboratory and synchrotron sources

X-ray microradiography is a well established technique for the study of biological structures in which the projected absorption is measured, usually with photographic film or resist. If scanning X-ray microradiography with a 15-μm beam, 2-D scanning, and photon counting is used, more accurate results can be obtained and real-time experiments undertaken. Addition of a rotation axis allows computerized axial tomography to be done at a resolution of 15 μm. This technique overcomes the inherent difficulty of microradiography that all detail perpendicular to the plane of the specimen is superimposed. This method has been applied to the study of the 3-D mineral distribution in a 0.8×0.8 mm column of human cortical bone with a laboratory X-ray source. Calculation of the wavelength dependence of the linear absorption coefficient for liver and bone shows that, for a choice of wavelength in the range of 3–0.4 Å (4–30 keV), the specimen thickness can be from 100μm–2 cm and 10 μm–3 mm, respectively. Synchrotron X-radiation has the potential for better resolution because of the higher intensity, which allows the use of a narrower beam. There is also the possibility of determining individual element 3-D distributions from measurements on either side of the absorption edges because of the continuous nature of the spectrum and also the possibility of doing this from X-ray fluorescence measurements. To investigate these possibilities, a tomographic apparatus has been built based on the availability of accurately ground, tungsten carbide balls. Metrological assessment shows that the specimen remains within <1 μm of the required position during translation and rotation. Preliminary X-ray tomographic studies with a 4-μm diameter beam have been started at the Daresbury laboratory synchrotron source.     

Syringe driver in terminal care.

Continuous subcutaneous infusions of drugs by syringe driver are used often and successfully in the terminal care of patients when drugs cannot be given orally. Diamorphine is the opioid of choice because of its high solubility. If other drugs such as antiemetics, anticholinergics, sedatives, or steroids are required they may also be given by syringe driver. This method is particularly useful for domiciliary care, where the practical difficulties of providing regular parenteral analgesia are otherwise formidable.     

Grb7 SH2 domain structure and interactions with a cyclic peptide inhibitor of cancer cell migration and proliferation

Background

Human growth factor receptor bound protein 7 (Grb7) is an adapter protein that mediates the coupling of tyrosine kinases with their downstream signaling pathways. Grb7 is frequently overexpressed in invasive and metastatic human cancers and is implicated in cancer progression via its interaction with the ErbB2 receptor and focal adhesion kinase (FAK) that play critical roles in cell proliferation and migration. It is thus a prime target for the development of novel anti-cancer therapies. Recently, an inhibitory peptide (G7-18NATE) has been developed which binds specifically to the Grb7 SH2 domain and is able to attenuate cancer cell proliferation and migration in various cancer cell lines.

Results

As a first step towards understanding how Grb7 may be inhibited by G7-18NATE, we solved the crystal structure of the Grb7 SH2 domain to 2.1 Å resolution. We describe the details of the peptide binding site underlying target specificity, as well as the dimer interface of Grb 7 SH2. Dimer formation of Grb7 was determined to be in the μM range using analytical ultracentrifugation for both full-length Grb7 and the SH2 domain alone, suggesting the SH2 domain forms the basis of a physiological dimer. ITC measurements of the interaction of the G7-18NATE peptide with the Grb7 SH2 domain revealed that it binds with a binding affinity of K_d = ~35.7 μM and NMR spectroscopy titration experiments revealed that peptide binding causes perturbations to both the ligand binding surface of the Grb7 SH2 domain as well as to the dimer interface, suggesting that dimerisation of Grb7 is impacted on by peptide binding.

Conclusion

Together the data allow us to propose a model of the Grb7 SH2 domain/G7-18NATE interaction and to rationalize the basis for the observed binding specificity and affinity. We propose that the current study will assist with the development of second generation Grb7 SH2 domain inhibitors, potentially leading to novel inhibitors of cancer cell migration and invasion.     

Arabinan-deficient mutants of Corynebacterium glutamicum and the consequent flux in decaprenylmonophosphoryl-D-arabinose metabolism

The arabinogalactan (AG) of Corynebacterianeae is a critical macromolecule that tethers mycolic acids to peptidoglycan, thus forming a highly impermeable cell wall matrix termed the mycolyl-arabinogalactan peptidoglycan complex (mAGP). The front line anti-tuberculosis drug, ethambutol (Emb), targets the Mycobacterium tuberculosis and Corynebacterium glutamicum arabinofuranosyltransferase Mt-EmbA, Mt-EmbB and Cg-Emb enzymes, respectively, which are responsible for the biosynthesis of the arabinan domain of AG. The substrate utilized by these important glycosyltransferases, decaprenylmonophosphoryl-D-arabinose (DPA), is synthesized via a decaprenylphosphoryl-5-phosphoribose (DPPR) synthase (UbiA), which catalyzes the transfer of 5-phospho-ribofuranose-pyrophosphate (pRpp) to decaprenol phosphate to form DPPR. Glycosyl compositional analysis of cell walls extracted from a C. glutamicum::ubiA mutant revealed a galactan core consisting of alternating beta(1-->5)-Galf and beta(1-->6)-Galf residues, completely devoid of arabinan and a concomitant loss of cell-wall-bound mycolic acids. In addition, in vitro assays demonstrated a complete loss of arabinofuranosyltransferase activity and DPA biosynthesis in the C. glutamicum::ubiA mutant when supplemented with p[14C]Rpp, the precursor of DPA. Interestingly, in vitro arabinofuranosyltransferase activity was restored in the C. glutamicum::ubiA mutant when supplemented with exogenous DP[14C]A substrate, and C. glutamicum strains deficient in ubiA, emb, and aftA all exhibited different levels of DPA biosynthesis.