Kathleen J. Barrett

Human ferritin H and L sequences lie on ten different chromosomes

SummaryIn humans, the H (heavy) and L (light) chains of the iron-storage protein ferritin, are derived from multigene families. We have examined the chromosomal distribution of these H and L sequences by Southern analysis of hybrid cell DNA and by chrosomal in situ hybridization. Our results show that human ferritin H genes and related sequences are found on at least seven different chromosomes while L genes and related sequences are on at least three different chromosomes. Further, we have mapped the chromosomal location of expressed genes for human H and L ferritin chains and have found an H sequence which may be a useful marker for idiopathic hemochromatosis.

High-specificity in-situ hybridization. Methods and application.

We describe a technique of in-situ hybridization using oligonucleotide probes employing the expression of immunoglobulin VH genes as a model. Optimal conditions for hybridization with the 35S-labeled oligonucleotide probes were established with monoclonal B-cell lines that express VH genes of known nucleic acid sequence. The range of sensitivity and specificity achieved with this technique is documented. Under conditions of high stringency. this method can detect the expression of highly related VH hypervariable regions.

A Prototype Anti‐DNA Autoantibody Appears to Define a New VH Gene That Is Conserved in Many Strains of Mice

A spontaneously arising monoclonal anti-DNA autoantibody derived from the lupusprone mouse strain, MRLllpr , defines a high frequency idiotype found in the sera of all MRLl lpr mice, but not in the sera of normal mice.’*’ We have cloned the two rearranged V, genes from hybridoma H130 and identified and sequenced the expressed V, gene (VH, D, and JH genes). This sequence is most closely related to that of 5558, a myeloma protein that binds ~~-1,3-dextran,~ and to two unexpressed VH genes, 108a and 108b, that were isolated from a BALB/c germline librarye4 The homology between these sequences a t the nucleic acid and amino acid levels is 86-90%. These four V regions differ from each other by 12-14 amino acids. The magnitude of this difference suggests that H130 is encoded by a new V, gene that has not yet been described; however, extensive somatic mutation cannot be excluded until germline genes have been sequenced. Hybridization a t high stringency to cloned H 130 probes that contained coding sequences, 5’-flanking sequences, or both, showed that the MLRllpr mouse has two EcoRI fragments that are highly homologous to the expressed V, gene from H 130 in both the coding and 5’-flanking regions. The same two fragments are conserved in normal and autoimmune strains that are haplotype j a t the Igh-V locus.536 Several strains of different haplotypes have at least one fragment that is highly homologous to the coding and flanking regions. These results suggest that the H130 V, gene or highly related V, genes are conserved in many strains of mice, but are not normally expressed.’ Rigorous proof of such a conclusion demands isolation and sequencing of these germline genes and examination of their expression. We have found that a panel of MRLl lpr anti-DNA autoantibodies are encoded by a minimum of ten different genes that belong to four of the known VH gene families. Some members of these four V, gene families might be especially well suited to code for anti-DNA autoantibodies. Thirty percent of these antibodies were encoded by members of the 7 183 family, which has been shown to be preferentially rearranged in pre-B The distribution is consistent with that found in immature B cells from adult bone marrow and neonatal day1 hybridoma~.’~~ However, the distribution of V genes expressed by this panel of autoantibodies could also be due to the stochastic use of V genes because of the size of the sample. Taken together, these results suggest that autoantibodies arise from multiple VH genes, either by direct activation of an immature population of B cells or by polyclonal activation of all B cells.