Cloned murine T200 (Ly-5) cDNA reveals multiple transcripts within B- and T-lymphocyte lineages.

Abstract

The murine T200 family of cell-surface glycoproteins is expressed on hematopoietic lineage cell types in a developmentally regulated manner with different lymphocyte subpopulations expressing cross-reactive but structurally distinct forms. To investigate the differences between these forms and the regulation of these differences, murine T200 cDNA clones were isolated by using a probe obtained by subtractive hybridization. This procedure made use of a T200' L-cell transfectant and the parent Ltk-cell line. The 1.9-kilobase (kb) cDNA clone was sequenced and found to contain the coding region of the COOH-terminal 331 amino acids and 0.9 kb of 3' untranslated region. Where the sequence overlapped with the rat sequence, 80-90% homology was observed. RNA blot analysis revealed that B-lineage cell lines express either a 5.6-kb or a 6.5-kb mRNA correlating to the size difference of the T200 glycoprotein synthesized. Similarly, in the T-cell lineage a helper T-cell clone and a cytotoxic T-cell clone express T200 transcripts of 5.6 kb and 5.9 kb, respectively, which correlate with the distinct sizes of their T200 glycoproteins. A T200-negative mutant of a T-cell line was found to express full-length T200 mRNA, although at a diminished level.

Isolation and Sequence of Partial cDNA Clones of Human L1: Homology of Human and Rodent L1 in the Cytoplasmic Region

“…A higher degree of sequence conservation in the cytoplasmic domain than in the extracellular domain has been seen previously in molecules such as T200 (Thomas et al, 1985;Ralph et al, 1987;Raschke, 1987) and the P-subunit of the fibronectin receptor (Tamkun et al, 1986;Argraves et al, 1987). It has been suggested that this conservation indicates an important functional role for the cytoplasmic domain, possibly in interacting with components in the cytoplasm or cytoskeleton.…”

ANALYSIS OF Ly5 CHROMOSOME 1 POSITION USING ALLELIC DIFFERENCES AND RECOMBINANT INBRED MICE

“…51. DNA marker sizes and sample origins are indicated; plasmid DNA samples of the BALB/c and SJA Ly5 molecule are shown and have been described previously (Raschke, 1987;Zebedee et al, 1991). observed previously in this laboratory, especially when probing membrane transfers with the oligonucleotide used in the PCR reactions, and may represent extended single strand DNA molecules. As these DNAs have not interfered with the Ly5 allele analysis, PCR conditions to eliminate them have not been devised.…”

“…Nucleotide sequence changes between cDNA clones of these two alleles have been examined by PCR techniques followed by restriction enzyme digestion (Zebedee el al., 1991). To characterize the Ly5 alleles among genomic DNA samples, a similar PCR-enzyme cleavage system was devised using information from the available Ly5 cDNA sequences (Saga et al, 1986;Raschke, 1987;Zebedee et al, 1991), the exon organization of the Ly5 gene (Saga et al, 1988), and allele-distinguishing restriction endonuclease sites (Fig. la).…”

“…As this incomplete digestion is observed in both cDNA and genomic lanes, the XhoI enzyme is not able to efficiently cleave amplified DNAs under the conditions employed. Nucleotide differences between the Ly5" allele and LySb allele were determined from cDNA sequences for SJL (Zebedee eral., 1991) and BALB/c (Saga etal., 1986;Raschke, 1987) LyS molecules. The nucleotide position and sequence changes between these alleles are shown with the corresponding alteration in DNA restriction enzyme site.…”

Genetic basis of antigenic differences between three alleles of Ly5 (CD45) in mice

“…The first half and last sixth of the extracellular domain are as conserved as the cytoplasmic domain. This region of the extracellular domain is also the region with the most variation between the rat and the mouse (Thomas et al 1985;Saga et al 1986;Barklay et al 1987;Raschke 1987). Although unlikely to cause major functional alterations, these differences do provide targets for allotypic immune responses.…”