Relationships between growth at the sutures of the rabbit calvarium

The present study was designed to elucidate the relationships between growth increments at the cranial vault sutures in rabbits. Thirteen male New Zealand white rabbits were followed regularly from age 31 to 142 days using a roentgen stereophotogrammetric system. Spherical tantalum markers were implanted into the nasal, frontal, and parietal bones, and implant stability was checked at each stereo examination. Problems with instability were encountered only in the nasal bones. Registered growth rates conformed to our previous investigations. High correlations were observed between the following areas; the coronal suture to the frontonasal suture, the first principal component of the neurocranial suture group to the frontonasal suture, and the principal component of the craniofacial suture group to the coronal suture. Remaining relationships demonstrated dispersion to various extents. The findings indicate that there seems to exist a basic mutual dependence between neural and facial skeletal growth, as well as complex covariations between the various sutures of the rabbit calvarium.     

The pericentriolar material in Chinese hamster ovary cells nucleates microtubule formation

The structure and function of the centrosomes from Chinese hamster ovary (CHO) cells were investigated by electron microscopy of negatively stained wholemount preparations of cell lysates. Cells were trypsinized from culture dishes, lysed with Triton X-100, sedimented onto ionized, carbon-coated grids, and negatively stained with phosphotungstate. The centrosomes from both interphase and dividing cells consisted of pairs of centrioles, a fibrous pericentriolar material, and a group of virus-like particles which were characteristic of the CHO cells and which served as markers for the pericentriolar material. Interphase centrosomes anchored up to two dozen microtubules when cells were lysed under conditions which preserved native microtubules. When Colcemid-blocked mitotic cells, initially devoid of microtubules, were allowed to recover for 10 min, microtubules formed at the pericentriolar material, but not at the centrioles. When lysates of Colcemid-blocked cells were incubated in vitro with micotubule protein purified from porcine brain tissue, up to 250 microtubules assembled at the centrosomes, similar to the number of microtubules that would normally form at the centrosome during cell division. A few microtubules could also be assembled in vitro onto the ends of isolated centrioles from which the pericentriolar material had been removed, forming characteristic axoneme- like bundles. In addition, microtubules; were assembled onto fragments of densely staining, fibrous material which was tentatively identified as periocentriolar material by its association of CHO can initiate and anchor microtubules both in vivo and in vitro.     

DNA gyrase is a host factor required for transposition of Tn5

We show that DNA gyrase is required for transposition of Tn5. Coumermycin, a potent inhibitor of DNA gyrase subunit B, inhibits transposition in a wild-type strain, but has no effect on strains carry ing a coumermycin-resistant allele in gyrB. In addition, strains containing a thermolabile subunit A of gyrase (gyrA43) are defective for transposition at a nonpermissive temperature. The requirement for gyrase is due to a requirement for supercoiled DNA. We showed this by introducing into the gyrA43 strain a deletion of the gene encoding topoisomerase I. The introduction of the second mutation caused an increase in the superhelical density of DNA as well as an increase in the transposition frequency. This also implies that if the DNA is supercoiled there is no further requirement for gyrase. Experiments with coumermycin support this, because the drug does not inhibit transposition if the recipient DNA remains supercoiled. This indicates that if the DNA acting as recipient of the transposon is deficient in supercoils, it will be a poor substrate for transposition. We also describe a system in which a gene on a multicopy plasmid can be efficiently introduced into the Escherichia coli chromosome.     

Transposition of the kanamycin-resistance transposon Tn903

Summary The insertion of the kanamycin-resistance transposon, Tn903, into the Escherichia coli chromosome was studied. Tn903 is similar in structure to the well known transposons Tn5 and Tn10 in that it has a unique central sequence flanked by inverted repeat sequences extending more than a thousand base pairs. However, the central region of Tn903 has enough single-frame coding capacity only for the drug modifying enzyme, whereas Tn5 and Tn10 carry multigenic unique sequences. In this paper we demonstrate that two different classes of insertion event occur: (1) the first class is a complex event in which all or part of the genome of the bacteriophage lambda vector is co-inserted near the purE locus on the E. coli chromosome (11.7 min); (2) the second class appears to be a simple transposition event in which the transposon alone is inserted at relatively nonspecific sites in the chromosome, as has been described for Tn5 and Tn10. Furthermore both classes show dependency on homology-requiring recombination systems. We suggest that Tn903 transposes infrequently because it must utilize a recA-controlled host function, whereas Tn5 and Tn10 are recA-independent and encode similar but more active functions on the transposon DNA.     

Conferral of transposable properties to a chromosomal gene in Escherichia coli

A normally stable gene of Escherichia coli was converted into a transposable element. A bacterial strain was constructed in which the malK gene was flanked on each side by the transposable element Tn5. The resulting Tn5-malK⁺-Tn5 structure (Tn651) became a transposable element with properties very similar to those of Tn5 itself. Tn651 transposes into regions of both the E. coli chromosome and bacteriophage lambda and is able to induce mutations. Transposition of Tn651 does not require the product of recA. Based on a physical analysis of lambda Tn651 DNA it is shown that the two Tn5s flanking the malK gene are in inverted orientation. In these experiments a new derivative of bacteriophage lambda is used that can accept a 14 kilobase insertion in vivo and still yield a plaque-forming transducing particle.     

Cdc42 interacts with the exocyst complex to promote phagocytosis

The process of phagocytosis in multicellular organisms is required for homeostasis, clearance of foreign particles, and establishment of long-term immunity, yet the molecular determinants of uptake are not well characterized. Cdc42, a Rho guanosine triphosphatase, is thought to orchestrate critical actin remodeling events needed for internalization. In this paper, we show that Cdc42 controls exocytic events during phagosome formation. Cdc42 inactivation led to a selective defect in large particle phagocytosis as well as a general decrease in the rate of membrane flow to the cell surface. Supporting the connection between Cdc42 and exocytic function, we found that the overproduction of a regulator of exocytosis, Rab11, rescued the large particle uptake defect in the absence of Cdc42. Additionally, we demonstrated a temporal interaction between Cdc42 and the exocyst complex during large particle uptake. Furthermore, disruption of exocyst function through Exo70 depletion led to a defect in large particle internalization, thereby establishing a functional role for the exocyst complex during phagocytosis.     

QTL mapping for two commercial traits in farmed saltwater crocodiles (Crocodylus porosus)

The recent generation of a genetic linkage map for the saltwater crocodile (Crocodylus porosus) has now made it possible to carry out the systematic searches necessary for the identification of quantitative trait loci (QTL) affecting traits of economic, as well as evolutionary, importance in crocodilians. In this study, we conducted genome‐wide scans for two commercially important traits, inventory head length (which is highly correlated with growth rate) and number of scale rows (SR, a skin quality trait), for the existence of QTL in a commercial population of saltwater crocodiles at Darwin Crocodile Farm, Northern Territory, Australia. To account for the uncommonly large difference in sex‐specific recombination rates apparent in the saltwater crocodile, a duel mapping strategy was employed. This strategy employed a sib‐pair analysis to take advantage of our full‐sib pedigree structure, together with a half‐sib analysis to account for, and take advantage of, the large difference in sex‐specific recombination frequencies. Using these approaches, two putative QTL regions were identified for SR on linkage group 1 (LG1) at 36 cM, and on LG12 at 0 cM. The QTL identified in this investigation represent the first for a crocodilian and indeed for any non‐avian member of the Class Reptilia. Mapping of QTL is an important first step towards the identification of genes and causal mutations for commercially important traits and the development of selection tools for implementation in crocodile breeding programmes for the industry.     

Growth of Legionella pneumophila in Dictyostelium discoideum: a novel system for genetic analysis of host-pathogen interactions

Legionella pneumophila, the Gram-negative bacterium that causes Legionnaires' disease, can be cultured in the laboratory in a variety of fresh-water amoebae and macrophage-like cell lines. None of these hosts, however, is amenable to genetic analysis, which has limited the ability of researchers to analyse the host factors essential for L. pneumophila growth. In this article, we describe a novel method in which L. pneumophila is grown within the soil amoeba Dictyostelium discoideum and how D. discoideum genetics is being used to analyse the host cell factors involved in L. pneumophila pathogenesis.     

Evidence for pore-forming ability by Legionella pneumophila

Legionella pneumophila is the cause of Legionnaires' pneumonia. After internalization by macrophages, it bypasses the normal endocytic pathway and occupies a replicative phagosome bound by endoplasmic reticulum. Here, we show that lysis of macrophages and red blood cells by L . pneumophila was dependent on dotA and other loci known to be required for proper targeting of the phagosome and replication within the host cell. Cytotoxicity occurred rapidly during a high-multiplicity infection, required close association of the bacteria with the eukaryotic cell and was a form of necrotic cell death accompanied by osmotic lysis. The differential cytoprotective ability of high-molecular-weight polyethylene glycols suggested that osmotic lysis resulted from insertion of a pore less than 3 nm in diameter into the plasma membrane. Results concerning the uptake of membrane-impermeant fluorescent compounds of various sizes are consistent with the osmoprotection analysis. Therefore, kinetic and genetic evidence suggested that the apparent ability of L . pneumophila to insert a pore into eukaryotic membranes on initial contact may play a role in altering endocytic trafficking events within the host cell and in the establishment of a replicative vacuole.