EXPRESSION OF H-2 ANTIGENS IN SJL/J TUMORS

Project: Research project

Description

SJL/J lymphomas can be used as a model of tumor progression from a
slow-growing malignancy to an aggressive tumor. We have previously shown
that one of the class I MHC antigens, D s , is not expressed in some SJL/J
lymphomas. The D s -negative tumors are not rejected and grow rapidly. The
goal of this research is to determine the structural defect(s) that
prevents expression of the D s antigens. Loss of expression could be due
to gross deletion or rearrangement of the gene or small alterations such as
point mutations or gene conversion. We will identify and sequence genomic
D s genes from normal tissue and from several malignant SJL/J lymphoma
lines. A comparison of the sequences will locate the gene(s). Since the
amino acid sequence of the D s glycoprotein is not known, the D s gene(s)
must be identified from the other 33-35 cross-hybridizing class I genes. A
combination of two approaches will be used to identify and determine the
number of D s genes: (1) several cDNA probes will be made from mRNA
isolated by immunopurification of polysomes. Size-selected,
double-stranded cDNA will be used for insertion into pBR322. These cDNA
clones will be tested for their ability to cross-hybridize with a class I
probe and then will be sequenced; and (2) the genomic D s gene(s) will be
identified from the other 33-35 cross-hybridizing class I genes on a
Southern blot by comparing the DNA from various H-2 congenic and intra-MHC
recombinant mouse strains as well as from many SJL/J lymphoma lines. The
D s gene(s) will be identified by its "unique" restriction enzyme site
polymorphism. This gene(s) will be cloned into a cosmid vector for
sequence analysis. The identify of the genomic D s gene(s) will be
confirmed by cloning the appropriate restriction fragment(s) containing the
D s gene(s) and comparing the sequence of the genomic gene(s) with the
D s cDNA sequence(s). The D s genes will be subcloned into the pSV2-neo
vector for both sequence and immunochemical analyses. Immunochemical
analyses of the glycoproteins produced in transformed mouse L cells will
confirm the production of clones containing the D s gene(s). Defective
D s genes will then be cloned from various SJL/J lymphoma lines and
sequenced. Such alterations in gene structure may be detected by
restriction site analyses on Southern blots followed by sequencing of the
appropriate segment of the tumor D s gene. These studies will, therefore,
identify the genetic mechanisms that prevent the expression of cell surface
antigens in malignancies. (AG)
StatusFinished
Effective start/end date7/1/8612/31/87

Funding

  • National Institutes of Health
  • National Institutes of Health

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H-2 Antigens
Genes
Neoplasms
MHC Class I Genes
Lymphoma
Complementary DNA
Glycoproteins
Gene Conversion
Cosmids
Histocompatibility Antigens Class I
Polyribosomes
Gene Rearrangement