FILTRADO Y UMBRALIZADO DE LA IMAGEN

The commensal lifestyle of the house m ouse is thought to have been adopted as a way to avoid competition w ith other m urids by habitat p artitio n in g ^^ . Ultimately, this life-style has led to the dom estication of the house m ouse, first as a pet and later as a laboratory animal. As, over time, this animal has becom e so im portant to genetidsts and developm ental biologists, the origin of the laboratory mice, the so-called "old inbred strains" has often been discussed.

They all have the sam e m tDNA haplotype, which is of M us musculus domesticus

origin ^89 Their nuclear genes in contrast show a higher level of divergence than expected given their recent derivation from a com m on ancestral sto c k ^ ^ , and this has led to speculation concerning the rate of evolution and m utation in these mice. It has been suggested that this divergence m ay be due to m uch higher rates of m utation occurring in the inbred lines, or that heterozygosity has been inadvertently selected for in the construction of the inbred lines by the selection of the m ost vigorous breeding pairs from each generation (to avoid inbreeding depression). A nother explanation stems from the finding that these strains all carry a Y chrom osom e of Mus musculus musculus o rig in ^ ^ , or m ore predsely, the variant only found in Japanese mice and som e Chinese mice2b9, 2^1. From the know n pedigrees of laboratory mice we know that, in addition to the know n inter-strain crosses that occurred early in their developm ent, occasional introductions of unknow n background have occurred, either by purchasing from specimens from pet dealers (Taney mice') or by crossing w ith animals trapped from the wüd. The presence of the Y chrom osom e of Asian origin confirms that the Oriental fancy mice has contributed to the ancestral stock of laboratory mice, through the early introduction of these types of mice (Japanese waltzing mice, for example) into European collections. In this way, the founder stock of the laboratory m ouse was a combination of very divergent genomes^57

1.8 Aims of the project

At the start of the project, Fvl activity had been isolated to a 6.5kb Spel-EcoRI fragm ent, b u t the gene itself had yet to be cloned. The retroviral nature of the gene had therefore not been determ ined, and it seem ed likely at this point that hom ologues of Fvl w ould exist in other species. The initial aim of this project was to isolate the hum an hom ologue of this gene and to see w hether the retroviral restrictive nature of the gene observed in mice existed, or could be induced through m utation, in hum ans. To this end, large-insert hu m an genomic libraries w ere screened for clones containing either or bo th of the hum an

hom ologues of the m urine genes that flanked Fvl in mice. From this clone(s) it

was hoped the h um an Fvl hom ologue could be subsequently isolated.

Once the Fvl gene in mice had been cloned, its retroviral nature was

dem onstrated by sequence hom ology to the HERV-L family of retroviruses.

Subsequently, Fvl was show n to be unique to m em bers of the M us genus only,

and the presence of hom ologues in other genera therefore seem ed highly unlikely. The aim of the project was then modified. By cloning and com paring Fvl sequences from m em bers across this genus, the next aim was to attem p t to determ ine how the gene had changed during the evolution of Mus, and to

ascertain how these changes related to the developm ent of Fvl activity in the

m ouse. To achieve this, the Fvl ORF was PCR-amplified and cloned from nearly

40 samples of m ouse genomic DNA. The Fvl consensus sequence was

determ ined for each sample, and these sequences w ere subjected to com puter- aided analysis.

C om plem entary to the sequencing project, I aim ed to clone a "progenitor^ viral

sequence of Fvl; the sequence of the gene shares hom ology w ith b o th the

FFERV-L family of retroviruses from hum ans and the MERV-L family of retroviruses from mice, b u t appears distinct from both. It was hoped that,

during such an infection that had given rise to the Fvl gene itself, another

germ line integration(s) had also occurred to fix a m ore complete example of this family of retrovirus in the m ouse genom e. In o rd er to isolate such a putative sequence(s) a m ouse genomic lam bda library was screened by hybridisation with

an Fvl probe at low stringency. Fragm ents that cross-hybridised w ith Fvl w ere

subcloned from the clones obtained from this screen, sequenced using prim ers flanking the cloning site, and these sequences w ere subsequently used to screen nucleic ad d sequence databases to identify them.

The final aim of the project w as to determ ine how the key changes betw een the m ain alleles related to Fvl activity. This data also related to the study of the

evolution of Fvl, as the sequencing project had show n how these changes had

occurred during the spéciation of Mus and the evolution of the Fvl phenotype.

Constructs containing m u tan t ORFs w ith the 3 m ain differences betw een FvV

and Fvl^ in all possible combinations w ere transfected in to the Fvl° cell line Mus

dunni. Fvl phenotype of clonal cell lines obtained from these transfections was

determ ined using a lacZ assay using pseudotyped MLV. The Fvl"^ allele was also

studied. Quantification of expression levels in tissue and sequence analysis of this allele was undertaken to determ ine w hat changes are likely to result in this modified phenotype.