The albino-deletion complex of the mouse: molecular mapping of deletion breakpoints that define regions necessary for development of the embryonic and extraembryonic ectoderm.

Previous complementation analyses with five (c11DSD, c5FR60Hg, c2YPSj, c4FR60Hd, c6H) of the mouse albino deletions defined at least two genes on chromosome 7, known as eed and exed, which are necessary for development of the embryonic and extraembryonic ectoderm, respectively, of early postimplantation embryos. The region of chromosome 7 containing these two genes has now been accessed at the molecular level by cloning two of the deletion breakpoint-fusion fragments. The c2YPSj breakpoints were isolated by cloning an EcoRI fragment containing a copy of an albino region-specific repeat unique to c2YPSj DNA. Similarly, the c11DSD breakpoints were isolated by cloning a c11DSD EcoRI fragment detected by a unique-sequence probe mapping proximal to the albino-coat-color locus. By mapping the cloned breakpoints relative to the remaining three deletions, the c11DSD distal breakpoint was found to define the distal limit of the region containing eed, whereas the c2YPSj and c6H distal breakpoints were found to define the proximal and distal limits, respectively, of the region containing exed.

Molecular cloning, expression, and chromosomal localization of a human gene encoding the CD33 myeloid differentiation antigen

Monoclonal antibodies of the CD33 cluster group recognize a 67- kilodalton (Kd) protein, designated p67, expressed on the surface of normal human myeloid progenitors and leukemic cells from most patients with acute myelogenous leukemia. The human gene encoding p67 was isolated in a mouse genetic background after DNA-mediated gene transfer and fluorescence-activated cell sorting (FACS) for transformants that bound the monoclonal antibody MY9. After three serial rounds of gene transfer and cell sorting, multiple independently derived tertiary mouse cell transformants were obtained that expressed p67. Southern blot analysis revealed that these transformants shared restriction fragments containing highly reiterated human DNA sequences. Two shared EcoRI fragments of 3.3-kilobase (kb) and 9.5-kb pairs were molecularly cloned into bacteriophage vectors. A subsegment of the 3.3-kb fragment lacking repeated sequences was then used as a unique sequence probe to isolate two independent cosmid clones. Cells transfected with DNA from both cosmid clones bound MY9, and the human p67 protein was demonstrated by immunoprecipitation. NFS mice inoculated with a mouse cell transformant coexpressing p67 and the v-fms oncogene product produced antisera that specifically immunoprecipitated p67 from human leukemic cell lines, mouse cell transformants, and mouse cells transfected with the biologically active cosmid clones. The human p67 locus was previously assigned to chromosome 19 by screening a panel of rodent X human somatic cell hybrids with the unique sequence probe. The gene was sublocalized to the q13.3 region of chromosome 19 by in situ hybridization. RNA transcripts of approximately 1.6 kb and 1.4 kb were identified in polyadenylated RNA from human myeloid leukemia cell lines using a probe from the genomic locus. Manipulation of the cloned p67 gene may provide insight into the function of its product and mechanisms regulating its expression.

Molecular cloning, expression, and chromosomal localization of a human gene encoding the CD33 myeloid differentiation antigen

Monoclonal antibodies of the CD33 cluster group recognize a 67- kilodalton (Kd) protein, designated p67, expressed on the surface of normal human myeloid progenitors and leukemic cells from most patients with acute myelogenous leukemia. The human gene encoding p67 was isolated in a mouse genetic background after DNA-mediated gene transfer and fluorescence-activated cell sorting (FACS) for transformants that bound the monoclonal antibody MY9. After three serial rounds of gene transfer and cell sorting, multiple independently derived tertiary mouse cell transformants were obtained that expressed p67. Southern blot analysis revealed that these transformants shared restriction fragments containing highly reiterated human DNA sequences. Two shared EcoRI fragments of 3.3-kilobase (kb) and 9.5-kb pairs were molecularly cloned into bacteriophage vectors. A subsegment of the 3.3-kb fragment lacking repeated sequences was then used as a unique sequence probe to isolate two independent cosmid clones. Cells transfected with DNA from both cosmid clones bound MY9, and the human p67 protein was demonstrated by immunoprecipitation. NFS mice inoculated with a mouse cell transformant coexpressing p67 and the v-fms oncogene product produced antisera that specifically immunoprecipitated p67 from human leukemic cell lines, mouse cell transformants, and mouse cells transfected with the biologically active cosmid clones. The human p67 locus was previously assigned to chromosome 19 by screening a panel of rodent X human somatic cell hybrids with the unique sequence probe. The gene was sublocalized to the q13.3 region of chromosome 19 by in situ hybridization. RNA transcripts of approximately 1.6 kb and 1.4 kb were identified in polyadenylated RNA from human myeloid leukemia cell lines using a probe from the genomic locus. Manipulation of the cloned p67 gene may provide insight into the function of its product and mechanisms regulating its expression.