GENETIC RECOMBINATION IN MURINE GERM CELLS 
Jan GELffiBTER, Ph.D., Assistant Investigator 
Dr. Geliebter and his colleagues are studying the 
molecular mechanisms by which multigene families 
evolve as a unit, a process referred to as concerted 
evolution. The laboratory is focusing on the role of 
recombination in the concerted evolution of the 
murine major histocompatibility complex (MHC). 
Histocompatibility molecules are the proteins 
found on the cells of mice and humans that are re- 
sponsible for transplant rejection. The biological 
function of these molecules is to bind viral, bacte- 
rial, and tumor antigens and present them to the 
immune system. The immune system can then at- 
tack and kill the infected cells and stop the spread 
of infection. Individual mice and humans have from 
three to six different histocompatibility molecules, 
each of which can bind a limited number of anti- 
gens. To bind a large number of these antigens and 
ensure the survival of the species, it is beneficial 
that many varieties of the histocompatibility mole- 
cules be found in the population. 
The three murine histocompatibility loci, K, D, 
and L, collectively make up the H-2 region of the 
murine MHC. The H-2 genes are members of the 
much larger class I multigene family of the MHC, 
which also includes the Qa and Tla region genes. 
Alleles of each H-2 locus exhibit high-sequence vari- 
ation (diversity) and are very polymorphic at the 
population level. Alleles of Qa and Tla region 
genes exhibit very little sequence diversity and are 
much less polymorphic than H-2 loci. Although the 
Qa and Tla region genes and their products share 
sequence homology and biochemical characteristics 
with the H-2 region genes and molecules, their bio- 
logical role is unknown. 
Mutant A* histocompatibility genes have been de- 
tected in C57BL/6 mice at the high frequency of ~1 
per 2,500 (alleles of H-2 loci from the C57BL/6 
strain are identified by a 6 superscript). Because 
they are histoincompatible with other members of 
the same inbred mouse strain, mice with mutant 
H-2 genes have been detected by skin grafting. Se- 
quence analyses have indicated that these mutant 
k'' genes contain clustered, multiple nucleotide al- 
terations. The mutant genes contain from two to 
seven nucleotide substitutions compared with the 
parental gene. Using oligonucleotide probes that 
are complementary to the mutant sequence in the 
altered gene. Dr. Geliebter has determined that 
the mutant k'' genes are generated by recombina- 
tion between the fd' gene and other class I genes. 
The mutant-specific oligonucleotide probes were 
hybridized to clones containing all of the class I 
genes of the C57BL/6 mouse, and upon sequenc- 
ing, the positive clones were found to contain the 
identical sequences as substituted into the mutant 
genes. 
These studies provided evidence that mutant 
histocompatibility genes arise via recombination 
events between K genes and other class I donor 
genes and result in the transfer of very small 
segments of DNA (<100 nucleotides) from donor 
genes to the gene. These very short genetic 
transfers have been termed microrecombinations. 
The microrecombinant genes are repetitive in 
nature; that is, the identical microrecombinant Kf' 
gene has been detected in independently arising 
mutant mice. It is thought that the products of 
many microrecombination events, accumulating in 
the K genes of a mouse population, result in 
the high-sequence diversity observed among K al- 
leles. 
Although it is certain that microrecombinations 
occur in germ cells of normal C57BL/6 mice, little is 
known about the molecular parameters of the 
microrecombination process. For example, it is not 
known whether microrecombinations proceed via a 
reciprocal (double crossover) or a nonreciprocal 
(gene conversion) mechanism. Furthermore, there 
is some genealogical evidence that microrecom- 
bination occurs only in female mice. In addition, 
microrecombinations may not occur in all strains of 
mice or with equal frequency in all H-2 genes of the 
same mouse. Further analyses of these aspects of 
the microrecombination process using classical skin 
graft techniques is either impractical or impossible. 
The emphasis of the research in Dr. Geliebter's 
laboratory is the analysis of the parameters and re- 
quirements of the microrecombination process. 
The approach being taken is the analysis of 
microrecombination products in murine germ cells; 
since microrecombinant mice come from micro- 
recombinant germ cells found in normal parents, 
the germ cells of normal mice should contain many 
microrecombinant A* genes (~1 in 5,000). In fact, 
since ovarian tissue from a single C57BL/6 fetus 
contains 25,000 germ cells (100,000 A* genes) 
there should be —20 microrecombinant A* genes 
per normal mouse. Since microrecombination 
events have been found to reoccur in nature, oligo- 
nucleotide probes for known microrecombinant 
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