An analysis of specific mouse liver cDNA clones

Several liver secretory protein cDNAs were isolated from a female BALB/c mouse liver cDNA library. The mouse major urinary proteins (MUPs) are encoded within a multigene family of about 35 genes. Most MUPs are members of either Group 1 or Group 2 sequences, which can be distinguished by DNA sequence divergence. Two of the sequenced clones, MUP 8 and MUP 11 were of the Group 1 type. A third MUP clone, MUP 15, has diverged from both Group 1 and Group 2 sequences (i.e. BS 6 and BS 2,3) by 15% and 17.4% respectively. The divergence is twice as great over exons 1-3 and the 3' terminal 68 nucleotides of the comparison, as it is over the intervening sequence. This suggests that an ancestral conversion event has occurred. MUP 15, like some Group 2 genes, has a longer signal peptide than Group 1 genes and differs from both Groups in having a probable N-linked glycosylation site and a different splice configuration between exons 6 and 7.Transferrin is the major iron binding protein in vertebrate serum. Transferrin cDNA clones corresponding to 1.16 Kb of the 3'half of the mRNA were isolated. The clones were identical where they overlapped, which implies that there is one predominantly expressed transferrin gene in mouse liver. Comparison of the mouse exonic sequence with human and chicken transferrins showed 18.0% and 35.5% replacement and 38.4% and 99.0% silent site divergence respectively. There are also small areas of higher homology within the domains, which may define iron binding sites. Preliminary investigations into two other cDNA clones are discussed. These correspond to the 3' end (950 Bp) of the third component of mouse complement and the N-terminal half, (810 Bp) of mouse contrapsin, which is homologous to human alpha^- antichymotrypsin

Interaction between p85 and Rab5 in the presences and absence of phosphorylated PDGFR peptide

The adaptor subunit of phosphatidylinositol 3'-kinases (PI3K), p85, is involved in many different biological processes. Recent studies have shown that one of these functions is to serve as a GTPase activating protein (GAP) towards Rab5, a small monomeric G-protein. Rab5, like other G-proteins, can bind to either GDP or GTP in vivo, assuming its inactive and active form, respectively. The p85 protein has been shown to associate with both the nucleotide-bound and nucleotide-free states of Rab5. It has also been shown that p85 associates with activated, phosphorylated platelet-derived growth factor receptors (PDGFRs) via its two SH2 domains, and that upon binding there is a conformational change in the p85 protein which leads to a derepression of p110 kinase activity. The purpose of this study was to analyze if binding of the activated PDGFR peptides to p85 affects its Rab5GAP activity, as well as to measure the binding affinity of p85 towards Rab5 in each of its nucleotide-bound states. GAP assays were performed to measure the effect that peptide analogs of both the activated and inactivated PDGFR had on p85 Rab5GAP activity, while the binding affinity of p85 towards Rab5 was measured using surface plasmon resonance. The results of this study suggest that PDGFR peptides have no significant effect on p85 Rab5GAP activity. Furthermore, p85 appears to have a higher magnitude of binding to nucleotide-associated Rab5 proteins, than nucleotide-free Rab5 proteins. It also appears that p85 forms more stable complexes with Rab5-GTP than with Rab5-GDP. These results further support previous studies that show p85 to be an important regulator of Rab5-mediated endosomal fusion and show that this activity is not regulated by binding to the activated PDGFR itself

Rox1 function in dosage compensation: structural / functional analysis of a non-coding rna

roX1 is a long non-coding RNA involved in the chromosome-wide gene regulation that occurs during dosage compensation in Drosophila. Dosage compensation in Drosophila melanogaster occurs by a global two-fold increase of transcription from the single male X chromosome. This essential process compensates for X chromosome monosomy. The male-specific lethal (MSL) complex, containing five proteins, localizes to the male X chromosome and alters chromatin to modify gene expression. roX1 and roX2 RNAs are redundant components of MSL complex that are required for its exclusive X-localization. Recent studies in our lab have revealed a second role of roX RNAs in heterochromatic gene expression in males. The roX-dependent heterochromatic regulation system involves some, but not all, MSL proteins. Although all components of this system discovered now are expressed in females, microarray analysis showed that the roX1 RNA has no detectable affect on expression of both X-linked and heterochromatic genes in females. Therefore, like dosage compensation, the roX-dependent heterochromatic regulation system is also limited to males. The differential regulation of heterochromatic genes in males and females may reflect the differences of heterochromatin between them. Previous studies of roX1 mutants and transgenes have identified a large region at the 5\u27 end of roX1 that is necessary for X-localization of the MSL complex. To dissect the function of this region, roX1 transgenes containing portions of the 5\u27 end were generated and analyzed. Multiple redundant elements contributing to X chromosome targeting were found to be present throughout the 5\u27 end of roX1. These roX1 transgenes display different stability, but all can partially restore X-linked gene expression in a roX1 roX2 mutant. One portion of this region is uniquely able to promote MSL complex spreading from sites of transcription. Previous model of MSL spreading suggested a verse relevance of roX1 transcript abundance and the ability to spread. However, the ability of this region to direct MSL spreading is not relevant to its abundance. The activities I have detected are hypothesized to be regulated by choice of transcription start site, alternative splicing and/or transcript stability

Regulation of Polarized Protein Transport to Axons, Dendrites, and Sensory Cilia in Caeborhabditis Elegans Neurons

Neurons are highly polarized cells, capable of receiving, processing and transmitting information, with the help of their specialized domains, an axon and one or more dendrites. The molecular dissimilarities between these domains are critical for neuronal function, and are a result of asymmetric trafficking of a large number of cellular components including ion channels, neurotransmitter receptors, synaptic vesicles, and signaling proteins. Yet, despite the significance of asymmetric protein transport in neuronal polarity, the molecules and mechanisms that direct polarized transport to axons, dendrites and cilia in neurons are only partly understood. In this thesis, I describe my effort at understanding how neuronal proteins are asymmetrically localized. I pursued a genetic approach, employing the C. elegans nervous system as an in vivo model system for protein transport to axons, dendrites and cilia. Having established a system to visualize axon-dendrite compartmentalization in PVD mechanosensory neurons, I identified the microtubule-binding protein UNC- 33/CRMP and the ankyrin homolog UNC-44 as major regulators of polarized protein transport in C. elegans neurons. In both unc-33 and unc-44 mutants, axonal proteins are distributed randomly between axons and dendrites, and dendritic proteins are partly mislocalized to axons. In both mutants, the axonal kinesin UNC-104/KIF1A actively misdelivers axonal proteins to both axons and dendrites. An altered distribution of neuronal microtubules in unc-33 and unc-44 mutants suggests that a primary defect in microtubule organization underlies defective protein targeting. unc-44 is required for UNC-33 localization to axons, where its enrichment in a proximal axonal segment suggests analogies with the vertebrate axon initial segment. In parallel experiments, I analyzed odr-8 mutants, which were previously identified in screens for chemotaxis-defective mutants and shown to affect GPCR localization. odr-8 mutants fail to localize a subset of odorant receptors including the ODR-10 diacetyl receptor to sensory cilia. I found that odr-8 encodes the C. elegans homolog of mammalian UfSP2, which acts as a cysteine-protease specific for UFM1, a ubiquitin-like molecule. ODR-10::GFP is retained in the ER in odr-8 mutants, whereas cilia localization of ODR-10::GFP is enhanced in ufm-1 mutants. ufm-1 function is required for ER retention of ODR-10::GFP in odr-8 animals. Thus, ODR-8 and UFM-1 may act antagonistically to regulate ER exit and cilia localization of chemoreceptors such as ODR-10

The Sequence of the Human Genome

A 2.91-billion base pair (bp) consensus sequence of the euchromatic portion of the human genome was generated by the whole-genome shotgun sequencing method. The 14.8-billion bp DNA sequence was generated over 9 months from 27,271,853 high-quality sequence reads (5.11-fold coverage of the genome) from both ends of plasmid clones made from the DNA of five individuals. Two assembly strategies—a whole-genome assembly and a regional chromosome assembly—were used, each combining sequence data from Celera and the publicly funded genome effort. The public data were shredded into 550-bp segments to create a 2.9-fold coverage of those genome regions that had been sequenced, without including biases inherent in the cloning and assembly procedure used by the publicly funded group. This brought the effective cov- erage in the assemblies to eightfold, reducing the number and size of gaps in the final assembly over what would be obtained with 5.11-fold coverage. The two assembly strategies yielded very similar results that largely agree with independent mapping data. The assemblies effectively cover the euchromatic regions of the human chromosomes. More than 90% of the genome is in scaffold assemblies of 100,000 bp or more, and 25% of the genome is in scaffolds of 10 million bp or larger. Analysis of the genome sequence revealed 26,588 protein-encoding transcripts for which there was strong corroborating evidence and an additiona

Portland Daily Press: December 29, 1896

https://digitalmaine.com/pdp_1896/1309/thumbnail.jp

Characterization of YisK, a Cell Shape Modifier and Enzyme in Bacillus subtilis

Bacterial growth and division requires the careful coordination of peptidoglycan (PG) synthesis and PG hydrolysis, allowing the insertion of new cell wall material at sites of active growth. In many rod-shaped bacteria, the bacterial actin homolog MreB is thought to coordinate this balance of synthesis and hydrolysis, particularly during cell elongation, and the current model is that MreB-like proteins act as a scaffold, directing the PG synthesis machinery to sites of active growth. Despite their importance, very little is known about how MreB-like proteins in prokaryotes are regulated. Using a Bacillus subtilis misexpression screen, we identified yisK and yodL, which cause a loss of cell shape and viability when misexpressed. Suppressors resistant to YisK’s killing activity primarily occur in mbl (the structural gene for an MreB paralog in B. subtilis), while suppressors resistant to YodL’s activity primarily occur in MreB. Consistent with the idea that YisK targets Mbl activity and YodL targets MreB activity, deletion of mbl confers resistance to YisK, while deletion of MreB confers resistance to YodL. In an mbl deletion background, YisK expressing cells also become 20% shorter, suggesting that YisK activity affects at least one other target integral to cell shape. Using a bacterial 2-hybrid assay, we detected an interaction between YisK and FtsE (the ATPase of the ABC Transporter FtsEX). Interestingly, published data indicates that FtsEX, which is important for regulating the activity of the D,L-endopeptidase CwlO, appears to act in the same pathway as Mbl, and both ftsE and cwlO mutants exhibit short-cell phenotypes. Our data suggest that ftsE is required for YisK-dependent cell shortening, but not cell widening. YisK shows ~40% amino acid identity to an FAH from Mycobacterium abcsessus, and we have obtained a preliminary crystal structure for YisK, with a dicarboxylic acid, most likely L-tartrate, bound in the active site. Surprisingly, introducing mutations in YisK’s active site has no effect on its ability to perturb cell shape. Our current model is that YisK is an enzyme, possibly involved in the dicarboxylate pathway, that utilizes interactions with Mbl and possibly FtsE to localize its enzymatic activity to specific regions within the cell

Strategy for an initial Measurement of the Inclusive Jet Cross Section with the CMS Detector

A strategy for an initial measurement of the inclusive jet cross section is presented and the related dominating systematic uncertainties are discussed. The study of this observable allows a fundamental probe of the theory of the strong interaction at unpreceeded energies. Additionally a method is presented to compare these measurements to calculations performed at next-to-leading order precision. In this context the dominating theoretical uncertainties are compared to the experimental ones