Understanding patterns of immunoglobulin gene rearrangements.

When immunoglobulin (Ig) gene rearrangements are analyzed, several patterns emerge. The rearrangements at the various loci generally appear in a specific temporal order. In addition, within a given locus the frequency of rearrangement of the various gene segments is not equal but is skewed towards preferential rearrangement of particular gene segments.

Conservation of sequence in recombination signal sequence spacers.

The variable domains of immunoglobulins and T cell receptors are assembled through the somatic, site specific recombination of multiple germline segments (V, D, and J segments) or V(D)J rearrangement. The recombination signal sequence (RSS) is necessary and sufficient for cell type specific targeting of the V(D)J rearrangement machinery to these germline segments. Previously, the RSS has been described as possessing both a conserved heptamer and a conserved nonamer motif.

Overusage of mouse DH gene segment, DFL16.1, is strain-dependent and determined by cis-acting elements.

The DJH structure is of particular importance for diversity in the immunoglobulin heavy chain because it encodes most of CDR3. Here, we investigate mechanisms responsible for generating the DJH structure. We found DFL16.

Map position and usage of 3' VH family members: usage is not position dependent.

The primary repertoire in mice, in large part, is determined by the Ig gene segments joined to form the variable region genes-VDJ for the heavy chain genes and VJ for the light chain genes. However, the mechanisms that determine which VH gene, of several hundred available, is joined to a DJH structure remain unexplained. One theory proposes that the VH gene segments closest to the 3' end of the VH locus are chosen because of their location, i.

High frequency of normal DJH joints in B cell progenitors in severe combined immunodeficiency mice.

The severe combined immunodeficiency (scid) mouse has a defective V(D)J recombinase activity that results in arrested lymphoid development at the pro-B cell stage in the B lineage. The defect is not absolute and scid mice do attempt gene rearrangement. Indeed, approximately 15% of all scid mice develop detectable levels of oligoclonal serum immunoglobulin and T cell activity.

B cell developmental defects in X-linked immunodeficiency.

We describe an analysis of early B cell development in mice with X linked immunodeficiency (Xid). It was found that, compared with the normal CBA/J strain, CBA/N (Xid/Xid) pre-B cells show an increased proliferative response to IL-7 but a decreased ability for subsequent maturation on stromal cells. However, the addition of mast cell growth factor largely restored the ability to mature in the presence of stromal cells.

Enumeration and characterization of DJH structures in mouse fetal liver.

The primary immunoglobulin (Ig) repertoire in the mouse develops during fetal life in the liver. The first Ig gene rearrangement--the joining of a DH to a JH gene segment--contributes largely to the diversity found in CDR3, as well as potentially encoding the D mu protein which is believed to function in the development of a B cell. In this report, the number of DJH joins in two mouse strains, C57BL/6 and BALB/c, were enumerated from days 12 to 16 of fetal development.

Mouse kappa light-chain recombination signal sequences mediate recombination more frequently than do those of lambda light chain.

Immunoglobulin and T-cell receptor genes are somatically rearranged by site-specific recombination. Recombination signal sequences (RSS) have been identified as the major targeting element of this process. Recent reports demonstrate that differences in RSS affect the frequency of recombination, suggesting a role for RSS in the development of the B-cell repertoire.

B cell commitment.

Multipotential stem cells have the capacity to differentiate into a variety of cell types including B lymphocytes. The development of B cells from multipotential progenitors has been the subject of numerous studies and many aspects of this transition are well characterized. However, the initial genetic events which commit a stem cell to follow the B lineage developmental pathway have not yet been discovered.

Measurement of recombinase activity in a set of related Abelson murine leukemic virus pre-B cell lines: DJ/DJ lines have more recombinase activity than do VDJ/VDJ lines.

The VDJ recombination potential of a number of Abelson murine leukemic virus transformed fetal liver cell lines derived from (C57BL/6 x BALB/c) F1 mice was measured. The specific developmental stage of each line was determined using Southern blot analysis to ascertain their rearrangement status at the immunoglobulin heavy chain locus (DJ/DJ, VDJ/DJ or VDJ/VDJ). It was observed that DNA from DJ/DJ lines gave many more 'subhaploid' bands hybridizing with JH than did DNA from VDJ/VDJ lines.

Ig gene rearrangements on individual alleles of Abelson murine leukemia cell lines from (C57BL/6 x BALB/c) F1 fetal livers.

We have previously shown that selection of Ig H chain V region genes used by colonies obtained from splenic B cells and fetal liver pre-B cells was dependent on strain-specific factors. Moreover, by examining the V gene usage in strains congenic at the Igh locus, we also determined that the strain-specific factor was encoded by sequences lying outside of the Igh locus. We decided to examine whether there are differences in Vh gene rearrangement between alleles in an F1 strain.

Association of Ig L chain-like protein lambda 5 with a 16-kilodalton protein in mouse pre-B cell lines is not dependent on the presence of Ig H chain protein.

Using a polyclonal rabbit antiserum against recombinant mouse lambda 5 protein, we determined that the pre-B cell specific mouse lambda 5 gene encodes a 22-kDa protein. The lambda 5 protein, which is related to conventional Ig lambda L chain proteins forms a complex with Ig mu H chain protein and an as yet unidentified 16-kDa protein (p16) in mu+ pre-B cell lines carrying a functionally rearranged VH-DH-JH allele. In pre-B cell lines which carry DH-JH rearrangements and do not express mu H chain protein, lambda 5 protein is associated with p16.

VH gene repertoire.

In this review, we have assembled some results on VH gene usage by mouse and human. We conclude that there is an early bias in usage of certain VH genes in both mouse and human. This biased usage has a strain dependent component as evidenced by its continued presence in the adult repertoire of some mouse strains, notably BALB/c, and not in others.

Characterization of a 70Z/3 pre-B cell derived macrophage clone. Differential expression of Hox family genes.

An adherent cell line was established from the much studied pre-B cell line 70Z/3. The adherent cell line had the morphological features of a macrophage and stained positive for non-specific esterase. In addition, this line expressed high levels of RNA for the macrophage specific enzyme, lysozyme.

The B cell repertoire.

The hallmark of the immune system is its ability to produce a seemingly infinite variety of antigen-binding receptors. This is made possible by molecular and cellular mechanisms uniquely suited to continuously generate a large number of individual receptor molecules and to select some for further expansion. The well-studied genetic rearrangement that results in the juxtaposition of germ line-encoded variable, diversity, and joining elements remains the foundation for diversification on which the repertoire is built.

A linkage map of the mouse immunoglobulin lambda light chain locus.

The mouse immunoglobulin lambda light chain locus has been linked using field inversion gel electrophoresis. The lambda light chain locus classically contains two V and four J-C gene segments in inbred mouse strains, and was physically mapped in the BALB/c cell line Wehi-3 which contains unrearranged lambda light chain gene segments. The locus is relatively small and spans 300 kb, as defined by a variety of single and double digests using methylation-sensitive restriction enzymes.

VH gene family utilization is regulated by a locus outside of the VH region.

We have used the RNA colony blot method to examine VH usage in colonies derived from primary splenic B cells. We found that there are strain-specific differences in the pattern of VH usage. Using parental F1, congenic, and recombinant inbred strains we demonstrate that the genetic element that causes the observed phenotype is: (a) stably expressed; (b) not due to maternal influence; (c) not due to dominate diffusible factors; (d) not linked to cloning efficiency; and (e) outside the Igh locus.

A sensitive dot blot procedure for detecting mRNA in lymphoid cells grown in liquid culture.

A sensitive dot blot procedure for detecting RNA transcripts with radiolabelled cDNA probes in small numbers of cells in suspension culture is described. For a frequent mRNA species such as that for immunoglobulin in an IgM secretor hybridoma, as few as 30 cells can be detected using a C mu probe; for a less frequent mRNA species such as transcripts coding for alpha, beta or gamma genes of the T cell receptor in T cell tumors or CTL clones, as few as 3 X 10(3) to 10(4) cells can be detected using C alpha, C beta or C gamma probes. High sensitivity is achieved by confining the cells to a very small area on the filter on which they are probed, and by treating the filter with formaldehyde.

Somatic point mutations in unrearranged immunoglobulin gene segments encoding the variable region of lambda light chains.

Somatic point mutations are usually found in the coding and flanking regions of functionally and aberrantly rearranged immunoglobulin variable region gene segments. Mutations in the unrearranged V gene segments of myelomas or hybridomas have not been described so far. We have cloned and sequenced unrearranged V lambda gene segments from several cell lines.