Rabbit Ig kappa 1b6 gene structure

Previous studies employing Southern blot analyses have detected multiple kappa-homologous sequences within EcoRI-digested DNA isolated from kappa 1b6 homozygous rabbits and kappa 1b6 L chain secreting RMH H158 cell line. These results are very unexpected because the published partial protein sequence for the kappa 1b6 C region is incompatible with an EcoRI restriction endonuclease recognition sequence at the nucleotide level for this allotype. To determine their identity, the kappa-homologous sequences were isolated from DNA extracted from a kappa 1b6 L chain secreting RMH H158 cell line by molecular cloning. Structural analyses demonstrated these sequences to contain genetic information encoding the majority of the kappa 1b6 L chain gene locus. The protein sequence deduced from the kappa 1b6 C region gene was shown to differ from the published partial kappa 1b6 C region protein sequence at five amino acid positions. One of these differences results in a glycine to serine interchange that introduces an EcoRI restriction endonuclease recognition site within the kappa 1b6 C region gene. Subsequent genomic Southern blot analyses confirmed this structural assignment. Based on these data, the EcoRI-sensitive kappa-homologous fragments present within the genomes of the RMH H158 cell line and kappa 1b6 homozygous rabbits represent the nominal kappa 1 gene and not an alternative kappa isotype or kappa pseudogene. Rabbit Ig kappa 1 allelic nucleotide sequence homology comparisons have shown the isolated kappa 1b6 J-C gene locus to display common structural features previously identified in other kappa 1 alleles.     

Differential fluorescence induction reveals Streptococcus pneumoniae loci regulated by competence stimulatory peptide

Differential fluorescence induction (DFI) in Streptococcus pneumoniae was used as a method for the discovery of genes activated in specific growth environments. Competence stimulatory peptide (CSP) was used as the model inducing system to identify differentially expressed genes. To identify CSP-induced promoters, a plasmid library was constructed by inserting random pieces of S. pneumoniae chromosomal DNA upstream of the promoterless gfpmut2 gene in an Escherichia coli/S. pneumoniae shuttle vector. S. pneumoniae carrying the library were induced with CSP and enriched for green fluorescent protein (GFP)-expressing bacteria using fluorescence-activated cell sorting. A total of 886 fluorescent clones was screened, and 12 differentially activated promoter elements were identified. Sequence analysis of these clones revealed that three were associated with novel competence loci, one of which we show is essential for DNA uptake, and six are known CSP-inducible promoters. We also explored whether competence proteins have a role in virulence and found that mutations in three CSP-inducible genes resulted in attenuated virulence phenotypes in either of two murine infection models. These results demonstrate the utility of DFI as a method for identifying differentially expressed genes in S. pneumoniae and the potential utility of applying DFI to other Gram-positive bacteria.     

IglC and PdpA Are Important for Promoting Francisella Invasion and Intracellular Growth in Epithelial Cells

The highly infectious bacteria, Francisella tularensis, colonize a variety of organs and replicate within both phagocytic as well as non-phagocytic cells, to cause the disease tularemia. These microbes contain a conserved cluster of important virulence genes referred to as the Francisella Pathogenicity Island (FPI). Two of the most characterized FPI genes, iglC and pdpA, play a central role in bacterial survival and proliferation within phagocytes, but do not influence bacterial internalization. Yet, their involvement in non-phagocytic epithelial cell infections remains unexplored. To examine the functions of IglC and PdpA on bacterial invasion and replication during epithelial cell infections, we infected liver and lung epithelial cells with F. novicida and F. tularensis ‘Type B’ Live Vaccine Strain (LVS) deletion mutants (ΔiglC and ΔpdpA) as well as their respective gene complements. We found that deletion of either gene significantly reduced their ability to invade and replicate in epithelial cells. Gene complementation of iglC and pdpA partially rescued bacterial invasion and intracellular growth. Additionally, substantial LAMP1-association with both deletion mutants was observed up to 12 h suggesting that the absence of IglC and PdpA caused deficiencies in their ability to dissociate from LAMP1-positive Francisella Containing Vacuoles (FCVs). This work provides the first evidence that IglC and PdpA are important pathogenic factors for invasion and intracellular growth of Francisella in epithelial cells, and further highlights the discrete mechanisms involved in Francisella infections between phagocytic and non-phagocytic cells.     

Detection of large cDNA inserts within crude lambda gt11 lysates: a rapid and sensitive method

A method is presented for the isolation of bacteriophage lambda DNA and the rapid identification of large cDNA inserts within crude phage lysates. The primary screening of a lambda gt11 cDNA library with a 32P-radiolabeled cDNA probe yielded 21 putative positive clones. A phage "spot-blot" analysis was employed to quickly screen these potential recombinants. This eliminated 9 of the 21 clones as the result of false positive signals. The remaining 12 recombinant phage were amplified on agarose-based media, and phage DNA was isolated using a modified plate lysate procedure. The DNA thus obtained from these crude lysates could be easily digested with EcoRI and examined by Southern blot analysis. The resulting blot was hybridized with the same cDNA probe used in the initial screening of the library. Thus, two clones harboring the longest cDNA insert were identified from a mixed phage population and were subsequently plaque purified. The procedure is rapid, sensitive, reproducible, inexpensive and allows the processing of several clones at once without sacrificing the quality or yields of the DNA preparation. Furthermore, the method obviates the need for plaque purifying all the positives obtained from the initial screening of a cDNA library.     

Molecular cloning and characterization of N-syndecan, a novel transmembrane heparan sulfate proteoglycan

A cDNA clone coding for a membrane proteoglycan core protein was isolated from a neonatal rat Schwann cell cDNA library by screening with an oligonucleotide based on a conserved sequence in cDNAs coding for previously described proteoglycan core proteins. Primer extension and polymerase chain reaction amplification were used to obtain additional 5' protein coding sequences. The deduced amino acid sequence predicted a 353 amino acid polypeptide with a single membrane spanning segment and a 34 amino acid hydrophilic COOH-terminal cytoplasmic domain. The putative extracellular domain contains three potential glycosaminoglycan attachment sites, as well as a domain rich in Thr and Pro residues. Analysis of the cDNA and deduced amino acid sequences revealed a high degree of identity with the transmembrane and cytoplasmic domains of previously described proteoglycans but a unique extracellular domain sequence. On Northern blots the cDNA hybridized to a single 5.6-kb mRNA that was present in Schwann cells, neonatal rat brain, rat heart, and rat smooth muscle cells. A 16-kD protein fragment encoded by the cDNA was expressed in bacteria and used to immunize rabbits. The resulting antibodies reacted on immunoblots with the core protein of a detergent extracted heparan sulfate proteoglycan. The core protein had an apparent mass of 120 kD. When the anti-core protein antibodies were used to stain tissue sections immunoreactivity was present in peripheral nerve, newborn rat brain, heart, aorta, and other neonatal tissues. A ribonuclease protection assay was used to quantitate levels of the core protein mRNA. High levels were found in neonatal rat brain, heart, and Schwann cells. The mRNA was barely detectable in neonatal or adult liver, or adult brain.     

Robust expansion of human hepatocytes in Fah-/-/Rag2-/-/Il2rg-/- mice

Mice that could be highly repopulated with human hepatocytes would have many potential uses in drug development and research applications. The best available model of liver humanization, the uroplasminogen-activator transgenic model, has major practical limitations. To provide a broadly useful hepatic xenorepopulation system, we generated severely immunodeficient, fumarylacetoacetate hydrolase (Fah)-deficient mice. After pretreatment with a urokinase-expressing adenovirus, these animals could be highly engrafted (up to 90%) with human hepatocytes from multiple sources, including liver biopsies. Furthermore, human cells could be serially transplanted from primary donors and repopulate the liver for at least four sequential rounds. The expanded cells displayed typical human drug metabolism. This system provides a robust platform to produce high-quality human hepatocytes for tissue culture. It may also be useful for testing the toxicity of drug metabolites and for evaluating pathogens dependent on human liver cells for replication.     

Real-time immuno-polymerase chain reaction in a 384-well format: Detection of vascular endothelial growth factor and epidermal growth factor-like domain 7

Immuno-polymerase chain reaction (immuno-PCR) combines the specificity of antibodies with the amplification power of PCR to detect low levels of proteins. Here, we describe the development of a 384-well immuno-PCR method that uses streptavidin coated on a PCR plate to capture complexes of biotinylated capture antibody, antigen, and DNA-labeled detection antibody. Unbound molecules are removed by a wash step using a standard plate washer. Antibody–DNA molecules in bound complexes are then detected directly on the plate using real-time PCR. Circulating human vascular endothelial growth factor concentrations measured by this method correlated with measurements obtained from enzyme-linked immunosorbent assay (ELISA). Using this method, we developed an assay for human epidermal growth factor-like domain 7 (EGFL7), an extracellular matrix-bound angiogenic factor. EGFL7 is expressed at a higher level in certain cancers, although endogenous EGFL7 concentrations have not been reported. Our 384-well EGFL7 immuno-PCR assay can detect 0.51 pM EGFL7 in plasma, approximately 16-fold more sensitive than the ELISA, utilizing the same antibodies. This assay detected EGFL7 in lysates of non-small-cell lung cancer and hepatocellular carcinoma cell lines and also hepatocellular carcinoma, breast cancer, and ovarian cancer tissues. This 384-well immuno-PCR method can be used to develop high-throughput biomarker assays.