Mouse chromosome 1

Chair of Committee for Mouse Chromosome 1     

Isogenic P-fimbrial deletion mutants of pyelonephritogenic Escherichia coli: the role of α Gal(1–4)β Gal binding in virulence of a wild-type strain

Escherichia coli strains causing acute pyelonephritis often express multiple fimbrial types and haemolysin, which may contribute to their ability to adhere to, and interact with, kidney epithelial cells. Strain CFT073, a pap⁺, sfa⁺, pil⁺, hly⁺ pyelonephritis strain, previously established as virulent in the CBA mouse model of ascending urinary tract infection and cytotoxic for cultured human renal epithelial cells, was selected for construction of isogenic strains. From a gene bank of this strain, two distinct copies of the pap operon were isolated. The two P-fimbrial determinants were sub-cloned into pCVD442, a positive selection suicide vector containing the sacB gene of Bacillus subtilis. Deletion mutations were introduced into each of the two constructs, within papEFG of one operon and papDEFG of the other. Suicide vectors carrying pap deletions were mobilized from E. coli SM10 lambda pir into CFT073 (Nal^R) and cointegrates were passaged on non-selective medium. The first pap mutation was identified by screening a Southern blot of DNA from sucrose-resistant colonies using a papEFG probe. This mutant retained the MRHA⁺ phenotype since a second functional copy of pap was still present. A double pap-deletion mutant, UPEC76, confirmed by Southern blotting, was unable to agglutinate human type O erythrocytes or α Gal(1–4)β Gal-coated latex beads. CBA mice (N =100) were challenged transurethrally with 10⁵, 10⁶, 10⁷, or 10⁹ cfu of strains CFT073 or UPEC76. After one week, quantitative cultures of urine, bladder, and kidney were done and histologic changes were examined. No substantive differences in organism concentration or histological findings between parent and mutant were detected in urine, bladder, or kidney at any challenge concentration. We conclude that adherence by P fimbriae of uro-pathogenic E. coli strain CFT073 plays only a subtle role in the development of acute pyelonephritis in the CBA mouse model.     

Cloning and molecular analysis of the galE gene of Neisseria meningitidls and its role in lipopolysaccharide biosynthesis

The galE gene from Haemophilus influenzae was used as a hybridization probe for the galE gene of Neisseria meningitidis Group B, identifying two different homologous loci. Each of the loci was cloned and nucleotide sequence analysis revealed that both loci contained sequences similar to galE. One contained a functional galE gene and mapped to the capsule biosynthetic locus. The second contained only a partial galE-coding sequence, which did not express a functional gene product. A galE mutant meningococcal strain was constructed by transformation with an inactivated galE gene. Analysis of the LPS from the galE mutant strain revealed an apparent reduction in molecular weight and a loss of reactivity with monoclonal antibodies specific for structures known to contain galactose. These results are consistent with an essential role for galE in the incorporation of galactose into meningococcal lipopolysaccharide.     

Effects of tRNALeu1 overproduction in Escherichia coli

Strains of Escherichia coli have been produced which express very high levels of the tRNA^leu₁ isoacceptor. This was accomplished by transforming cells with plasmids containing the leuV operon which encodes three copies of the tRNA^Leu₁ gene. Most transformants grew very slowly and exhibited a 15-fold increase in cellular concentrations of tRNA^Leu₁ As a result, total cellular tRNA concentration was approximately doubled and 56% of the total was tRNA^Leu₁. We examined a number of parameters which might be expected to be affected by imbalances in tRNA concentration: in vivo tRNA charging levels, misreading, ribosome step time, and tRNA modification. Surprisingly, no increase in intracellular ppGpp levels was detected even though only about 40% of total leucyl tRNA was found to be charged in vivo. Gross ribosomal misreading was not detected, and it was shown that ribosomal step times were reduced between two- and threefold. Analyses of leucyl tRNA isolated from these slow-growing strains showed that at least 90% of the detectable tRNA^Leu₁ was hypomodified as judged by altered mobility on RPC-5 reverse-phase columns, and by specific modification assays using tRNA(m¹G)-methyltransferase and pseudo-uridylate synthetase. Analysis of fast-growing revertants demonstrated that tRNA concentration per se may not explain growth inhibition because selected revertants which grew at wild-type growth rates displayed levels of tRNA comparable to that of control strains bearing the leuV operon. A synthetic tRNA^Leu₁ operon under the control of the T7 promoter was prepared which, when induced, produced six- to sevenfold increases in tRNA^Leu₁ levels. This level of tRNA^Leu₁ titrated the modification system as judged by RPC-5 column chromatography. Overall, our results suggest that hypomodified tRNA may explain, in part, the observed effects on growth, and that the protein-synthesizing system can tolerate an enormous increase in the concentration of a single tRNA.     

Deletion analysis of the SUP35 gene of the yeast Saccharomyces cerevisiae reveals two non-overlapping functional regions in the encoded protein

SUP35is an omnipotent suppressor gene of Saccharomyces cerevisiae coding for a protein consisting of a C-terminal part similar to the elongation factor EF-1α and a unique N-terminal sequence of 253 amino acids. Twelve truncated versions of the SUP35 gene were generated by the deletion of fragments internal to the coding sequence. Functional studies of these deletion mutants showed that: (i) only the EF-1α-like C-terminal part of the Sup35 protein is essential for the cell viability; (ii) overexpression of either the N-terminal part of the Sup35 protein or the full-length Sup35 protein decreases translational fidelity, resulting in omnipotent suppression and reduced growth of [psi⁺] strains; (iii) expression of the C-terminal part of the Sup35 protein generates an antisuppressor phenotype; and (iv) both the N- or C-terminal segments of the Sup35 protein can bind to 80S ribosomes. Thus, the data obtained define two domains within the Sup35 protein which are responsible for different functions.     

Characterization of anf genes specific for the alternative nitrogenase and identification of nif genes required for both nitrogenases in Rhodobacter capsulatus

To identify Rhodobacter capsulatus nif genes necessary for the alternative nitrogenase, strains carrying defined mutations in 32 genes and open reading frames of nif region A, B or C were constructed. The ability of these mutants to grow on nitrogen-free medium with molybdenum (Nif phenotype) or in a nifHDK deletion background on medium without molybdenum (Anf phenotype) was tested. Nine nif genes and nif-associated coding regions are absolutely essential for the alternative nitrogenase. These genes comprise nifV and nifB, the nif-specific ntr system (nifR1, R2, R4) and four open reading frames, which exhibit no homology to known genes. In addition, a significantly reduced activity of both the alternative nitrogenase and the molybdenum-dependent nitrogenase was found for fdxN mutants. By random Tn5 mutagenesis of a nifHDK deletion strain 42 Anf^? mutants were isolated. Southern hybridization experiments demonstrated that 17 of these Tn5 mutants were localized in at least 13 different restriction fragments outside of known nif regions. Ten different Anf^? Tn5 mutations are clustered on a 6 kb DNA fragment of the chromosome designated anf region A. DNA sequence analysis revealed that this region contained the structural genes of the alternative nitrogenase (anfHDGK). The identification of several Tn5 insertions mapping outside of anf region A indicated that at least 10 genes specific for the alternative nitrogenase are present in R. capsulatus.     

Phylogeny of Saxifraga section Saxifraga subsection Arachnoideae (Saxifragaceae) and the origin of low elevation shade-dwelling species

Saxifraga section Saxifraga subsection Arachnoideae is a lineage of 12 species distributed mainly in the European Alps. It is unusual in terms of ecological diversification by containing both high elevation species from exposed alpine habitats and low elevation species from shady habitats such as overhanging rocks and cave entrances. Our aims are to explore which of these habitat types is ancestral, and to identify the possible drivers of this remarkable ecological diversification. Using a Hybseq DNA-sequencing approach and a complete species sample we reconstructed and dated the phylogeny of subsection Arachnoideae. Using Landolt indicator values, this phylogenetic tree was used for the reconstruction of the evolution of temperature, light and soil pH requirements in this lineage. Diversification of subsection Arachnoideae started in the late Pliocene and continued through the Pleistocene. Both diversification among and within clades was largely allopatric, and species from shady habitats with low light requirements are distributed in well-known refugia. We hypothesize that low light requirements evolved when species persisting in cold-stage refugia were forced into marginal habitats by more competitive warm-stage vegetation. While we do not claim that such competition resulted in speciation, it very likely resulted in adaptive evolution.     

A fast computational model for circulatory dynamics: effects of left ventricle–aorta coupling

The course of diseases such as hypertension, systolic heart failure and heart failure with a preserved ejection fraction is affected by interactions between the left ventricle (LV) and the vasculature. To study these interactions, a computationally efficient, biophysically based mathematical model for the circulatory system is presented. In a four-chamber model of the heart, the LV is represented by a previously described low-order, wall volume-preserving model that includes torsion and base-to-apex and circumferential wall shortening and lengthening, and the other chambers are represented using spherical geometries. Active and passive myocardial mechanics of all four chambers are included. The cardiac model is coupled with a wave propagation model for the aorta and a closed lumped-parameter circulation model. Parameters for the normal heart and aorta are determined by fitting to experimental data. Changes in the timing and magnitude of pulse wave reflections by the aorta are demonstrated with changes in compliance and taper of the aorta as seen in aging (decreased compliance, increased diameter and length), and resulting effects on LV pressure–volume loops and LV fiber stress and sarcomere shortening are predicted. Effects of aging of the aorta combined with reduced LV contractile force (failing heart) are examined. In the failing heart, changes in aortic properties with aging affect stroke volume and sarcomere shortening without appreciable augmentation of aortic pressure, and the reflected pressure wave contributes an increased proportion of aortic pressure.

     

Insulin-like growth factors: Putative muscle-derived trophic agents that promote motoneuron survival

Treatment of chick embryos in ovo with IGF-I during the period of normal, developmentally regulated neuronal death (embryonic days 5–10) resulted in a dose-dependent rescue of a significant number of lumbar motoneurons from degeneration and death. IGF-II and two variants of IGF-I with reduced affinity for IGF binding proteins, des(1-3) IGF-I and long R³ IGF-I, also elicited enhanced survival of motoneurons equal to that seen in IGF-I-treated embryos. IGF-I did not enhance mitogenic activity in motoneuronal populations when applied to embryos during the period of normal neuronal proliferation (E2-5). Treatment of embryos with IGF-I also reduced two types of injury-induced neuronal death. Following either deafferentation or axotomy, treatment of embryos with IGF-I rescued approximately 75% and 50%, respectively, of the motoneurons that die in control embryos as a result of these procedures. Consistent with the survival-promoting activity on motoneurons in ovo, IGF-I, -II, and des(1-3) IGF-I elevated choline acetyltransferase activity in embryonic rat spinal cord cultures, with des (1-3) IGF-I demonstrating 2.5 times greater potency than did IGF-I. A single addition of IGF-I at culture initiation resulted in the maintenance of 80% of the initial ChAT activity for up to 5 days, during which time ChAT activity in untreated control cultures fell to 9%. In summary, these results demonstrate clear motoneuronal trophic activity for the IGFs. These findings, together with previous reports that IGFs are synthesized in muscle and may participate in motoneuron axonal regeneration and sprouting, indicate that these growth factors may have an important role in motoneuron development, maintenance, and recovery from injury. © 1993 John Wiley & Sons, Inc.