Evaluating the importance of the different sources of variations is essential in microarray data experiments. Complex experimental designs generally include various factors structuring the data which should be taken into account. The objective of these experiments is the exploration of some given factors while controlling other factors. We present here a family of methods, the analyses with respect to instrumental variables, which can be easily applied to the particular case of microarray data. An illustrative example of analysis with instrumental variables is given in the case of microarray data investigating the effect of beverage intake on peripheral blood gene expression. This approach is compared to an ANOVA-based gene-by-gene statistical method. Instrumental variables analyses provide a simple way to control several sources of variation in a multivariate analysis of microarray data. Due to their flexibility, these methods can be associated with a large range of ordination techniques combined with one or several qualitative and/or quantitative descriptive variables.
Analysis of TCR-beta gene recombination and expression was performed by quantitative PCR amplification technique throughout chicken embryogenesis and development. Our data demonstrated that TCR V beta 1 promoters were turned on by day 10 of embryogenesis, 2 days before detection of TCR-beta gene recombination. The V to D recombination step was first detected by day 11 of embryogenesis whereas DJ and V(D)J rearranged genes were detected 1 day later, on day 12 of embryogenesis. Thus, transcription of unrearranged TCR-beta genes in chickens precedes the expression of V(D)J recombinase activity as in mammals. In contrast, although TCR-beta rearrangement starts with the D to J recombination step in mammals, it can start either by the VD or the DJ step in chickens. Furthermore, reverse transcriptase-PCR amplification of TCR-beta transcripts revealed the presence of two kinds of alternative transcripts. These novel alternatively spliced products appeared in thymocytes from embryonic thymus during colonization periods and were absent in transformed T cell lines. Splicing sites are located in the middle of V beta 1 segments and lead to delta V beta 1-C beta and delta V beta 1-D beta-J beta-C beta transcripts. delta V beta 1-C beta transcripts might lead to synthesis of invariant truncated TCR beta-chains containing the aminoterminal portion of the V beta 1 region followed by the C beta region. Because this type of splicing can be generated by using all known V beta 1 members, these invariant forms could play a role in thymocyte development.
Among the rheumatoid factors (RFs), monospecific and polyspecific types can be distinguished. However the molecular basis responsible for their different specificity is not well understood. In a previous report, we have shown that the binding of the majority of the polyspecific antibodies is salt-sensitive. No binding to IgG was observed under high ionic strength (0.3-0.5 M NaCl). This salt-sensitivity was only observed for 18% of the monospecific RFs. Here, we have analyzed 14 RFs representing the 3 different groups (6 salt-insensitive monospecific, 4 salt-sensitive monospecific and 4 salt-sensitive polyspecific RFs). By analysis of the amino acid composition and the distribution of polar and non-polar residues of their heavy chain complementarity-determining region 3 (H-CDR3) in relation to mono/polyspecifieity, salt-sensitivity and reactivity against human IgG subclasses, we have identified common structural features responsible for their different binding properties. Salt-sensitive RFs (mono as well as polyspecific antibodies) were characterized by long H-CDR3′s (15.3 ± 2.7) that contained large numbers of hydrophilic residues such as arginine and serine, while salt-insensitive RFs had more hydrophobic H-CDR3′s of smaller length (11.3 ± 2.4). In addition, for the monospecific RFs, remarkably similar hydrophilicity H-CDR3 profiles were found that were correlated with their specificity for IgG subclasses. These observations confirm the importance of the H-CDR3 for the binding of RFs to IgG. Furthermore, on the basis of their shorter H-CDR3′s and their rather unique H-CDR3 hydrophilicity profiles, it is likely that the majority of the monospecific RFs should be considered as a group of RFs that is independent of the polyspecific RF repertoire.
