LAMBDA LIGHT CHAIN IMMUNOGLOBULIN GENES

Project: Research project

Project Details

Description

A long-term goal of this project is to better understand the molecular
basis of immunoglobulin (Ig) gene rearrangement and expression. The human
Ig lambda light chain gene complex offers two advantages: it is a system
where the molecular regulation of DNA expression of closely linked, very
similar genes may be studied. In addition, lambda accounts for about 40% of
human serum Igs and therefore study of its expression is of importance for
further knowledge of the human immune system. Lambda light chains occur as
one of four isotypes which are expressed in human myeloma proteins in the
percentages: 15%Mcg, 61% Ke-Oz-, 18% Ke-Oz+ and 6%Ke+Oz-. In addition to
these genes for serum proteins the lambda locus encodes gene products
expressed only early in B cell ontogeny (14.1, 16.1 and our new 16.2, which
are equivalent to C-lambda-5 in mouse) which may be involved in B cell
development. This proposal is studying the expression and the molecular
regulation of the expression of these human C-lambda genes.

This project will complete the characterization (sequence of the human
lambda constant region genes. It will use isotype-specific probes to
determine the total level of expression of the particular lambda isotypes
(Mcg, Ke-Oz-, Ke-Oz+ encoded respectively by genes C-lambda-1, 2, 3 in
various cell compartments (blood, lymphnode, spleen) and the fraction of
lambda human hybridomas (HuHy) with the particular isotypes. It will also
determine the representation of the newest C-lambda gene, C-lambda-7, which
we have recently isolated and sequenced, in the above cell populations. The
lambda genes 14.1, 16.1 and 16.2 the last being one which we have cloned
and sequenced, will also be studied in similar fashion. The techniques to
be used include the polymerase chain reaction (PCR) on primer extensions
(cDNA) of lambda mRNA and hybridization to excess isotype-specific
oligonucleotides. The level of nascent transcripts (run-on transcription),
steady state level and RNA half life of the lambda isotypes in permanent
cell lines (HuHy) and normal lymphoid populations will be determined.
Potential transcriptional enhancer sequences will be located by
transfecting permanent lymphoid lines (both lambda-producing and non-
producing) via electroporation with CAT (chloramphenicol acetyl
transferase) vectors containing our test sequence. Differential enhancer
activity will be measured for lambda gene segments and will allow us to
test the hypothesis that increased enhancer function is correlated with
increased gene rearrangement and hence gene expression. The DNase
hypersensitivity of rearranged and unrearranged gene segments will be
determined which will help us localize potential enhancer regions and will
establish whether both homologous chromosomes are in an open configuration.
StatusFinished
Effective start/end date12/31/894/30/96

Funding

  • National Institute of Allergy and Infectious Diseases
  • National Institute of Allergy and Infectious Diseases
  • National Institute of Allergy and Infectious Diseases
  • National Institute of Allergy and Infectious Diseases
  • National Institute of Allergy and Infectious Diseases
  • National Institute of Allergy and Infectious Diseases
  • National Institute of Allergy and Infectious Diseases
  • National Institute of Allergy and Infectious Diseases
  • National Institute of Allergy and Infectious Diseases

Fingerprint

Explore the research topics touched on by this project. These labels are generated based on the underlying awards/grants. Together they form a unique fingerprint.