Identification and gene organization of three novel members of the IL-1 family on human chromosome 2.

Members of the IL-1 family of cytokines are important in mediating inflammatory responses. The genes encoding IL-1alpha, IL-beta, and the IL-1 receptor antagonist (IL-1Ra) are clustered within 450 kb on human chromosome 2q. By searching the EST databases and sequencing this region of chromosome 2, we have identified three novel genes that show homology to the IL-1 family, which we have named IL-1-related protein 1, 2, and 3 (IL-1RP1, IL-1RP2, and IL-1RP3). All three genes contain a signature motif common to the IL-1 family and appear to be more closely related to IL-1Ra. Similar to the intracellular form of IL-1Ra, these genes lack conventional hydrophobic signal sequences. The expression of these genes appears to be highly restricted to various epithelial cell populations. Our results demonstrate the existence of additional IL-1 gene family members within the previously defined IL-1 cluster and point to this region of chromosome 2 as an evolutionary hotspot for IL-1 gene duplication. These genes may prove to have an important role in inflammatory responses.

Chromosomal localization and genomic characterization of the mouse melastatin gene (Mlsn1).

We recently described a novel gene, melastatin, whose expression is inversely correlated with melanoma aggressiveness. Chromosomal localization of this gene places it on mouse chromosome 7 and in the 15q13-q14 region of the human genome. Although expression patterns and chromosomal localization in the mouse are consistent with involvement of melastatin mutations in the mouse ruby-eye-2 defect, congenic analysis showed genetic segregation of the two loci. Cloning of the full-length human cDNA revealed a much larger transcript than we had previously identified, corresponding to a 1533-amino-acid protein product with homology to members of the transient receptor potential (Trp) family of calcium channels. The mouse melastatin gene contains 27 exons and spans at least 58 kb of genomic DNA. The promoter region of Mlsn1 contains four potential microphthalmia binding sites including an M box, a transcriptional regulatory element unique to genes with a restricted melanocytic expression pattern. A 1-kb PvuII fragment from this region was capable of driving high levels of luciferase expression in B16 melanoma cells.

Theoretical and empirical issues for marker-assisted breeding of congenic mouse strains.

Congenic breeding strategies are becoming increasingly important as a greater number of complex trait linkages are identified. Traditionally, the development of a congenic strain has been a time-consuming endeavour, requiring ten generations of backcrosses. The recent advent of a dense molecular genetic map of the mouse permits methods that can reduce the time needed for congenic-strain production by 18-24 months. We present a theoretical evaluation of marker-assisted congenic production and provide the empirical data that support it. We present this 'speed congenic' method in a user-friendly manner to encourage other investigators to pursue this or similar methods of congenic production.

The physical and genetic map surrounding the Lyst gene on mouse chromosome 13.

During the recent cloning of the mouse Lyst gene we developed both a high-resolution genetic map and a complete YAC and BAC contig of the Lyst critical region on mouse Chromosome 13. We also report the mapping of the human homologue of the mouse Lyst gene (LYST) to 1q43. These data are consistent with LYST being the gene for the human Chediak-Higashi Syndrome and strengthen the synteny relationship between MMU13 and human 1q43.

Identification and expression cloning of a leptin receptor, OB-R.

The ob gene product, leptin, is an important circulating signal for the regulation of body weight. To identify high affinity leptin-binding sites, we generated a series of leptin-alkaline phosphatase (AP) fusion proteins as well as [125I]leptin. After a binding survey of cell lines and tissues, we identified leptin-binding sites in the mouse choroid plexus. A cDNA expression library was prepared from mouse choroid plexus and screened with a leptin-AP fusion protein to identify a leptin receptor (OB-R). OB-R is a single membrane-spanning receptor most related to the gp130 signal-transducing component of the IL-6 receptor, the G-CSF receptor, and the LIF receptor. OB-R mRNA is expressed not only in choroid plexus, but also in several other tissues, including hypothalamus. Genetic mapping of the gene encoding OB-R shows that it is within the 5.1 cM interval of mouse chromosome 4 that contains the db locus.

Genetic analysis of colon cancer susceptibility in mice.

Mouse models may aid in the identification of genes involved in colon cancer. Our mating scheme involved mouse strains selected for maximum differences in susceptibility to DMH-induced colon tumors. Tumors were found in 40 of 122 progeny from a backcross to the resistant strain. We examined progeny animals for segregation of 177 genetic markers distributed at intervals of 5-30 cM on all mouse chromosomes. Multiple loci contribute to the phenotype, with significant linkage to a novel locus, Ccs1, between D12Mit5 and D12Mit6 on mouse Chr 12. Comparative maps suggest that the human homologue of Ccs1 is near FOS on human chromosome 14q.

Isolation and partial characterization of the Campylobacter rectus cytotoxin.

Previously, we reported the antigranulocytic activity of Campylobacter rectus media supernatants containing lipopolysaccharide (LPS) and a 104 kDa protein. Here, we monitored the release of protein and LPS through the growth cycle of C. rectus ATCC 33238 and identified the 104 kDa protein as the cytotoxin. LPS in media supernatants was quantitated by a KDO assay; the 104 kDa protein was detected on immunoblots with specific antibody (A104) and quantitated by amino acid analysis of membrane immobilized protein bands. C. rectus cell product release was independent of cell lysis. Over 24 h, the 104 kDa protein was released linearly while LPS was released in two plateaus; both increased in C. rectus culture supernatants 3 h after inoculation achieving maximum concentrations at 21 h of 3.1 micrograms/ml and 14.6 micrograms/ml, respectively. In 2 h, trypan blue viability assays, 37-47 micrograms of 12, 18 and 24 h supernatant protein killed 33-43% of HL-60 cells. Supernatant toxicity was heat sensitive and inhibited by A104. Sequencing the 16 N-terminal amino acids of the cytotoxin distinguished it from described C. rectus proteins. Similarities between epitopes and amino acid compositions of the Actinobacillus actinomycetemcomitans leukotoxin and C. rectus cytotoxin were observed. These data indicate that C. rectus secretes a 104 kDa cytotoxin.

Production of an extracellular toxin by the oral pathogen Campylobacter rectus.

The ATCC type strain and six clinical isolates of Campylobacter rectus were tested for toxicity against HL-60 cells and human polymorphonuclear neutrophils (PMNs). After challenge with bacterial cell suspensions and media supernatants for up to 4 h, eukaryotic cell viability was assayed by trypan blue dye exclusion and lactate dehydrogenase release. Cells of the C. rectus type strain were not toxic. However, ethanol and (NH4)2SO4 extracts of culture media supernatants killed HL-60 cells in a time and dose dependent manner with 700 micrograms of supernatant protein killing 100% of HL-60 cells in 4 h. Concentrated media supernatants from clinical isolates also killed 100% of HL-60 cells in 30 to 60 min. The bacterial culture supernatants were toxic to PMNs with clinical isolates killing 70 to 90% of PMNs in 2 to 4 h. SDS-PAGE and immunoblot analysis of the toxic media supernatants revealed C. rectus specific proteins and lipopolysaccharide (LPS). The toxic activity was inhibited by protease, indicating that the toxin was protein. Non-toxic and toxic media supernatants were obtained by altering hemin and fumarate in the growth media. SDS-PAGE analysis of these revealed that all toxic supernatants contained a 104 kDa protein.

Molecular genetic analysis of Actinobacillus actinomycetemcomitans epidemiology.

Research over the past decade has identified many of the microorganisms involved in the etiology of human periodontitis such as Actinobacillus actinomycetemcomitans. Efforts are now directed toward defining these species' role in the pathogenic process. Since microbial colonization of host tissues is a key first step in developing a bacterial infection, determining the source of the periodontal pathogens and their route of transmission is likely to be crucial in formulating preventive strategies. Recently, a technique from molecular biology, restriction endonuclease analysis, has been used to track bacterial infections. In the present study, this method was used to investigate the epidemiology of A. actinomycetemcomitans infection. One hundred twenty-four human subgingival plaque isolates of A. actinomycetemcomitans were examined including bacterial strains from the United States, Korea, and Norway as well as 15 strains from cynomolgus (Macaca fascicularis) and spider monkeys (Macaca iris) and 4 reference strains. The genomic DNA from each strain was purified, digested with each of 16 restriction endonucleases, and the DNA digests were resolved by electrophoresis. The resulting patterns of DNA fragments were compared and also correlated with the A. actinomycetemcomitans serotype determined using serotype-specific antisera in immunofluorescence. Human isolates of A. actinomycetemcomitans even from disparate geographic sources showed little diversity by restriction endonuclease analysis. Three major restriction patterns were found. Restriction pattern I was common to all 20 of the serotype a isolates, restriction pattern II was associated with 58% of the 73 serotype b isolates examined, while restriction pattern III was associated with the remaining serotype b strains and with all 15 of the serotype c strains.(ABSTRACT TRUNCATED AT 250 WORDS)