Nonrandom location of IS1 elements in the genomes of natural isolates of Escherichia coli

We have studied the spatial distribution of IS1 elements in the genomes of natural isolates comprising the ECOR reference collection of Escherichia coli. We find evidence for nonrandomness at three levels. Many pairs of IS1 elements are in much closer proximity (< 10 kb) than can be accounted for by chance. IS1 elements in close proximity were identified by long-range PCR amplification of the genomic sequence between them. Each amplified region was sequenced and its map location determined by database screening of DNA hybridization. Among the ECOR strains with at least two IS1 elements, 54% had one or more pairs of elements separated by < 10 kb. We propose that this type of clustering is a result of "local hopping," in which we assume that a significant proportion of tranposition events leads to the insertion of a daughter IS element in the vicinity of the parental element. A second level of nonrandomness is found in strains with a modest number of IS1 elements that are mapped through the use of inverse PCR to amplify flanking genomic sequences: in these strains, the insertion sites tend to be clustered over a smaller region of chromosome than would be expected by chance. A third level of nonrandomness is observed in the composite distribution of IS elements across strains: among 20 mapped IS1 elements, none were found in the region of 48-77 minutes, a significant gap. One region of the E. coli chromosome, at 98 min, had a cluster of IS1 elements in seven ECOR strains of diverse phylogenetic origin. We deduce from sequence analysis that this pattern of distribution is a result of initial insertion in the most recent common ancestor of these strains and therefore not a hot spot of insertion. Analysis using long- range PCR with primers for IS2 and IS3 also yielded pairs of elements in close proximity, suggesting that these elements may also occasionally transpose by local hopping.      

Relationship between longevity of spermatozoa after insemination and the percentage of normal embryos in brown marsupial mice (Antechinus stuartii)

Twenty-six female brown marsupial mice in a laboratory colony were mated at intervals ranging from 1 to 20 days between coitus and ovulation. The numbers of corpora lutea and normal embryos were counted. A multiple regression model examined the parabolic relationship between the proportion of normal embryos and the time from coitus to ovulation. The proportion of normal embryos increased until a mean of 9.5 days and decreased thereafter. This relationship was independent of the year of breeding and the number of corpora lutea. After survival of spermatozoa for up to 13 days in the female reproductive tract, the fertility levels of females was 88-92%. Low fertility levels after 13 days appeared to be due to a decrease in the number of spermatozoa. Reproductive tracts from 7 females killed after insemination and examined histologically showed many spermatozoa in the isthmus of the oviduct and the uterus at 5 days post coitum; spermatozoa confined to the isthmus between 6 and 13 days; and few spermatozoa in the isthmus at 14 days after copulation. A comparison between the fertility levels in the females which had been inseminated once and a further 17 females which had been inseminated 2 or 3 times suggested that spermatozoa from 2nd and 3rd inseminations can contribute spermatozoa for fertilization. In these females fertility levels did not decline with time after the first mating.     

One-proton catalysis by the alpha-L-arabinofuranosidase III of Monilinia fructigena.

1. Michaelis-Menten parameters for the hydrolysis of p-nitrophenyl alpha-L-arabinofuranoside were measured as a function of pL (pH or pD) in both 1H2O and 2H2O. 2. The variation of both Vmax. and Vmax./Km with pL is sigmoid, the pK governing Vmax. shifting from 6.34 +/- 0.05 in 1H2O to 6.84 +/- 0.07 in 2H2O, and that governing Vmax./Km from 5.89 +/- 0.03 in 1H2O to 6.38 +/- 0.05 in 2H2O. 3. In the plateau regions there is a small inverse solvent isotope effect on Vmax./Km (0.92), and one of 1.45 on Vmax. 4. The variation of Vmax. with isotopic composition is strictly linear, indicating that the isotope effect arises from the transfer of a single proton.     

Fitness effects of amino acid replacements in the beta-galactosidase of Escherichia coli

Two genetic procedures were used to obtain amino acid replacements in the lacZ-encoded beta-galactosidase in Escherichia coli. Amino acid replacements could be obtained without regard to their effects on lactase activity by selecting spontaneous mutations that relieved the strong polarity of six nonsense mutations. When streaked on MacConkey- lactose indicator plates, approximately 75% of these mutants gave strong red lactose-fermenting colonies, and 25% gave white nonfermenting colonies. Mutants from 11 other nonsense codons were isolated directly using MacConkey-lactose indicator plates, on which positive color indication requires only 0.5% of the wildtype lactase activity. Among the total of 17 codons, 25 variant beta-galactosidases were identified using electrophoresis and thermal denaturation studies. The fitness effects of these variant beta-galactosidases were determined using competition experiments conducted with lactose as the sole nutrient limiting the growth rate in chemostat cultures. Three of the replacements were deleterious, one was selectively advantageous, and the selective effects of the remaining 21 were undetectable under conditions in which the smallest detectable selection coefficient was approximately 0.4%/generation.      

The mechanism of cellular orientation during early cartilage formation in the chick limb and regenerating amphibian limb

A characteristic feature of early cartilage formation is that as the cells at the centre differentiate into chondroblasts they become flattened at right angles to the long axis of the cell mass, whereas the cells in the region of the future perichondrium become flattened circumferentially. On the basis of EM observation a simple model is proposed which is based on matrix secretion flattening the cells.     

Dynamic dissociating homo-oligomers and the control of protein function

Homo-oligomeric protein assemblies are known to participate in dynamic association/disassociation equilibria under native conditions, thus creating an equilibrium of assembly states. Such quaternary structure equilibria may be influenced in a physiologically significant manner either by covalent modification or by the non-covalent binding of ligands. This review follows the evolution of ideas about homo-oligomeric equilibria through the 20th and into the 21st centuries and the relationship of these equilibria to allosteric regulation by the non-covalent binding of ligands. A dynamic quaternary structure equilibria is described where the dissociated state can have alternate conformations that cannot reassociate to the original multimer; the alternate conformations dictate assembly to functionally distinct alternate multimers of finite stoichiometry. The functional distinction between different assemblies provides a mechanism for allostery. The requirement for dissociation distinguishes this morpheein model of allosteric regulation from the classical MWC concerted and KNF sequential models. These models are described alongside earlier dissociating allosteric models. The identification of proteins that exist as an equilibrium of diverse native quaternary structure assemblies has the potential to define new targets for allosteric modulation with significant consequences for further understanding and/or controlling protein structure and function. Thus, a rationale for identifying proteins that may use the morpheein model of allostery is presented and a selection of proteins for which published data suggests this mechanism may be operative are listed.     

Cloning and characterization of a new gene from the PAT protein family,in a marsupial,the stripe‐faced dunnart (Sminthopsis macroura)

Recent studies of PAT proteins in Drosophila and Xenopus have revealed significant roles for this family of proteins in the polarized transport of lipid droplets and maternal determinants during early embryogenesis. In mammals, PAT proteins are known to function mainly in lipid metabolism, yet research has yet to establish a role for PAT proteins in mammalian embryogenesis. Oocytes and early cleavage stages in Sminthopsis macroura show obvious polarized cytoplasmic distribution of organelles, somewhat similar to Drosophila and Xenopus, suggesting that a PAT protein may also be involved in S. macroura embryonic development. In the present study, we identified a new marsupial gene for PAT family proteins, DPAT, from S. macroura. Expression analyses by RT‐PCR and whole mount fluorescent in situ hybridization revealed that DPAT expression was specific to oocytes and cleavage stage conceptuses. Analysis of the localization of lipid droplets during S. macroura early embryonic development found a polarized distribution of lipid droplets at the two‐ and four‐cell stage, and an asymmetric enrichment in blastomeres on one side of conceptuses from two‐ to eight‐cell stage. Lipid droplets largely segregate to pluriblast cells at the 16‐cell stage, suggesting a role in pluriblast lineage allocation. Mol. Reprod. Dev. 77: 373–383, 2010. © 2010 Wiley‐Liss, Inc.     

Tuftsin signals through its receptor neuropilin‐1 via the transforming growth factor beta pathway

Tuftsin (Thr‐Lys‐Pro‐Arg) is a natural immunomodulating peptide found to stimulate phagocytosis in macrophages/microglia. Tuftsin binds to the receptor neuropilin‐1 (Nrp1) on the surface of cells. Nrp1 is a single‐pass transmembrane protein, but its intracellular C‐terminal domain is too small to signal independently. Instead, it associates with a variety of coreceptors. Despite its long history, the pathway through which tuftsin signals has not been described. To investigate this question, we employed various inhibitors to Nrp1's coreceptors to determine which route is responsible for tuftsin signaling. We use the inhibitor EG00229, which prevents tuftsin binding to Nrp1 on the surface of microglia and reverses the anti‐inflammatory M2 shift induced by tuftsin. Furthermore, we demonstrate that blockade of transforming growth factor beta (TGFβ) signaling via TβR1 disrupts the M2 shift similar to EG00229. We report that tuftsin promotes Smad3 phosphorylation and reduces Akt phosphorylation. Taken together, our data show that tuftsin signals through Nrp1 and the canonical TGFβ signaling pathway.

     

A timetable of embryonic development, and ovarian and uterine changes during pregnancy, in the stripe-faced dunnart, Sminthopsis macroura (Marsupialia: Dasyuridae)

Aged stages (63) were available for establishment of a timetable of embryonic development of the stripe-faced dunnart. On Day 0 oocytes reaching maturity were found in the ovary. Within +/- 24 h of time 0 (time of minimum morning weight) polymorphonuclear leucocytes appeared and spermatozoa were last detected in the urine of 70% of females. Embryos were collected at intervals during pregnancy by hemihysterectomy and the embryos in the contralateral uterus either were examined at a later stage of pregnancy or allowed to develop to term. Cleavage to the unilaminar blastocyst stage with around 32 cells took 3 days with a cleavage arrest of 24 h at the 4-cell stage. Expansion of the unilaminar blastocyst occurred over the next 3 days. Primitive endoderm cells appeared on Day 6, fully bilaminar blastocysts by the end of Day 7 and trilaminar blastocysts on Day 8. Shell loss and implantation of 13-15-somite stage embryos occurred on Day 8 and organogenesis over the next 2-3 days. The gestation period was 9.5-12.0 days with most births occurring between 10.5 and 11.0 days. Major steps in embryonic development were correlated with stages in the development of the corpora lutea, which were maximal in size, and possibly in secretory activity, when the embryos were at the bilaminar blastocyst stage. Regression commenced when the embryos were at the primitive streak stage. At the time the corpora lutea were maximal the uterine epithelium reached its greatest height and the endometrium was thick and folded. Later in pregnancy villous-like projections of the epithelium formed, and the luminal epithelial cells became rounded. Two cell populations, a tier of 8 smaller cells above the yolk mass and a tier of 8 larger cells around the sides of the yolk mass appeared at the 16-cell stage. From the 16-cell stage to the blastocyst stage, with 150-200 cells, two cell populations distinguished by size, cell cycle time, cytoplasmic appearance and position relative to the yolk mass were present. The two populations were indistinguishable in blastocysts with greater than 200 and less than 2000 cells. They reappeared in blastocysts with greater than 2000 cells, as the darker cells of the embryoblast, and as the paler cells of the trophoblast. The darker cells lay in the yolky hemisphere and the paler cells in the non-yolky hemisphere.