Each V gene begins with sequence encoding a signal peptide of about 22 amino acids. The general structures of the germline V genes are similar for the three Ig loci: heavy chain, k, and l. In many myelomas, both chromosomes are present and both are rearranged.Ī more complete understanding of recombination of Ig genes developed from sequence analysis of cloned myeloma versus germline DNA. The germline-sized fragments (hatched bands in the Southern blots) may or may not be preserved in the myeloma, depending on whether the nonexpressed homologous chromosome has remained in its germline (unrearranged) state. In myeloma DNA ( lower panel), V and C genes have been brought into close proximity and, in this example, are no longer separated by EcoRI sites both genes are found on the same EcoRI fragment of 6 kb, which is thus identified by either probe. The V region probe hybridizes to a family of related genes (shown by bands above and below the 3-kb band). In germline DNA ( upper drawings), V and C are an unknown distance apart and are found by Southern blot hybridization ( left) to lie on EcoRI fragments of 3 and 5 kb, respectively. EcoRI sites in this hypothetical example are indicated by arrows. Southern blot demonstration of rearrangement of immunoglobulin V and C region genes. This result contradicted the possibility that allelic exclusion might be explained by a mechanism that allowed recombination on only one chromosome, and it raised questions about the nature of the "second" gene rearrangement in these cells, as discussed later in this chapter.įIG. In panels of myelomas analyzed for C k recombination by Southern blotting, many showed evidence of DNA rearrangement on both allelic chromosomes. Results like these for k and l genes strongly supported the Dreyer-Bennett hypothesis and forcefully challenged the concept that every cell in the body has identical genes ( 2, 3). Similarly, one of the V region bands may be expected to be rearranged in the myeloma so as to lie on a different-sized fragment, the same fragment that hybridizes to the C k probe. The new rearranged band may be larger, smaller, or, fortuitously, the same size as the germline band, depending on the location of the restriction sites flanking the V and C genes. 1, the recombination bringing a V gene close to a C gene can cause an alteration in size of the C k-hybridizing restriction fragment. The hypothesis that recombination occurs between V and C genes is supported by the different bands observed when these probes are hybridized to myeloma DNA instead of germline DNA. These observations support the multiple V genes, single C gene component of the Dreyer-Bennett model. 1, probes representing different expressed V k genes are found to hybridize to a different set of bands, representing a different family of related V k genes. Moreover, although this is not shown in Fig. A probe representing an expressed V k gene detects several bands, as though hybridizing to a family of related sequences. 1 for an analysis of k light-chain genes, a C k probe detects only a single band in germline DNA, consistent with a single C k gene. Myeloma and germline DNA were initially compared by Southern blotting with hybridization probes derived from myeloma complementary DNAs (cDNAs). OVERVIEW OF IMMUNOGLOBULIN V GENE ASSEMBLYĮvidence from Southern Blotting and Gene Cloning Chapter 5 Immunoglobulins: Molecular Genetics
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