Gene expression profiling following BMP-2 induction of mesenchymal chondrogenesis in vitro.

OBJECTIVE: This study aims to apply gene expression profiling technology to gain insight into the molecular regulation of mesenchymal chondrogenesis. METHODS: The experimental system consists of micromass cultures of C3H10T1/2 cells, a murine multipotential embryonic cell line, treated with the chondroinductive growth factor, bone morphogenetic factor-2 (BMP-2). In this system, chondrogenic differentiation characterized by both morphological changes and cartilage matrix gene expression has been shown to be completely dependent upon BMP-2 treatment and the high cell plating density of micromass cultures. To identify candidate genes that may have key functional roles in chondrogenesis, we have applied subtractive hybridization to isolate genes whose expression is significantly up- or down-regulated during chondrogenesis. RNA was isolated from micromass cultures treated with BMP-2 for 24 h and analysed for representational differences by means of a subtractive hybridization screening method. RESULTS: Sixteen different genes were identified whose expression was up-regulated between two- and 12-fold by B,P-2, and twelve different genes were identified whose expression was down-regulated between two- and seven-fold by BMP-2. CONCLUSIONS: The potential of this screening methodology to identify new BMP-2 regulated genes is suggested by the fact that a majority of the identified genes are indeed novel. Identification and characterization of these genes should provide insight as to how chondrogenesis is regulated and also should provide important new markers for the study of osteoarthritis.

Contrasting molecular epidemiology of group A streptococci causing tropical and nontropical infections of the skin and throat.

Disease caused by group A streptococci (GAS) in tropical regions often takes the form of impetigo, whereas pharyngitis tends to predominate in temperate zones. GAS derived from asymptomatic throat infections and pyoderma lesions of rural Aboriginal Australians were evaluated for phylogenetic distant emm genes, which represent ecological markers for tissue site preference. On the basis of the percentage of total isolates from a given tissue, emm pattern A-C organisms exhibited a stronger predilection for the throat, whereas pattern D organisms preferred the skin. Only 16% of isolates collected by active surveillance displayed pattern A-C, which reflects the low incidence of oropharyngeal infection. Importantly, most (70%) pattern A-C organisms were isolated from skin sores, despite their innate tendency to infect the throat. Combined with findings from nontropical populations, analysis of the data supports the hypothesis that GAS tissue preferences are genetically predetermined and that host risk factors for infection strongly influence the differential reproduction of individual clones.

The ZF87/MAZ transcription factor functions as a growth suppressor in fibroblasts.

ZF87/MAZ is a zinc finger transcription factor that activates expression of tissue-specific genes and represses expression of the c-myc proto-oncogene. Infection of NIH3T3 fibroblasts with a retrovirus expressing ZF87/MAZ leads to a significant reduction in G418-resistant colonies, compared to cells infected with a retroviral control. Further, only a small fraction of the G418-resistant colonies express ZF87/MAZ. When the ZF87/MAZ-expressing colonies are expanded, they demonstrate a slow growth phenotype, a delayed transit through G1 phase and a decrease in endogenous c-myc gene expression and cyclin A and cyclin E protein levels. Consistent with a partial G1 arrest, the ZF87/MAZ-expressing cells show a reduced sensitivity to the S phase specific chemotherapeutic agent camptothecin. These data indicate that ZF87/MAZ is a growth suppressor protein in nontransformed cells, in part, by affecting the levels of key cell cycle regulatory proteins.

Transcriptional repression from the c-myc P2 promoter by the zinc finger protein ZF87/MAZ.

ZF87/MAZ is a zinc finger-containing transcription factor that was cloned based on its ability to bind to a site within the c-myc P2 promoter. However, its role in the control of c-myc transcription has not yet been well established. Here we have analyzed the effect of ZF87/MAZ overexpression on transcription from the murine c-myc P2 promoter. It was found that when overexpressed in COS cells, ZF87/MAZ significantly represses transcription from P2. The repression is mediated through the ME1a2 element, located at position -86 relative to the P2 transcriptional start site, and is not mediated through either the E2F or the ME1a1 sites. ZF87/MAZ functions as a true transcriptional repressor since it can repress transcription independently of the c-myc promoter, as part of a fusion with the GAL4 protein. The repressive domain within ZF87/MAZ is located in the amino-terminal half of the protein, a region rich in proline and alanine residues. ZF87/MAZ therefore shares features (i.e. a Pro/Ala-rich region) with those of known transcriptional repressor proteins.

Genetic linkage of exotoxin alleles and emm gene markers for tissue tropism in group A streptococci.

In group A streptococci, genetic markers for principal tissue reservoir are located within emm genes, which encode surface proteins that have a role in virulence. A worldwide collection of 160 isolates was evaluated for two traits: chromosomal emm gene markers for tissue tropism (designated patterns A-E), and bacteriophage-associated genes (speA and speC) encoding pyrogenic exotoxins. The speA and speC alleles of organisms harboring the emm marker for a pharyngeal reservoir (pattern A-C) differ from spe alleles that predominate in organisms with the emm marker for impetigo (pattern D). However, organisms that display the emm marker for both tissue sites (pattern E) are not intermediate for the distribution of either speA or speC alleles, but instead resemble pattern A-C isolates for speA and pattern D strains for speC. Statistically significant nonrandom associations between exotoxin alleles and emm patterns were observed but cannot be readily explained by niche separation alone.

Two-domain motif for IgG-binding activity by group A streptococcal emm gene products.

A biological role for the non-immune binding of human IgG by group A streptococci is evidenced by its strong association with a subpopulation of strains giving rise to tissue-specific infection. IgG-binding activity lies within many of the M and M-like surface proteins (encoded by emm genes), and several structurally distinct IgG-binding sites are known to exist. In this report, two adjacent IgG-binding domains, differing in their specificity for human IgG subclasses, are localized within the M-like protein, protein H. The putative coding regions for the two IgG-binding domains were mapped for 82 epidemiologically unrelated strains. Both coding regions are associated with phylogenetically distant emm genes, supporting a role for horizontal transfer and intergenomic recombination in the evolution of emm genes. In most instances, the two coding regions are tightly linked, suggesting that there exist strong selective pressures to maintain a two-domain binding motif. Both coding regions are found among all strains bearing emm gene markers associated with impetigo lesions as the principal tissue reservoir, but are absent from most strains that exhibit markers for a predominant nasopharyngeal reservoir. The data support the hypothesis that the pathogenic potential of an isolate is dictated, at least in part, by its unique array of multifunctional emm gene products.