Molecular cloning and exon-intron mapping of the gene encoding human transmembrane secretory component (the poly-Ig receptor)

Secretory component (SC or the poly-Ig receptor) plays a crucial role in mucosal immunity by translocating polymeric IgA and IgM through secretory epithelial cells into external body fluids. Labeled restriction fragments from human SC cDNA were used to screen a human genomic leukocyte library. Three overlapping clones, spanning a total of 19 kb of the human SC gene, including 3 kb of the 5' flanking region, were characterized. The putative TATA box candidate, preceded by a CAAT-like box, was found 329 nucleotides upstream of the first exon. Altogether 11 exons covering the entire coding region were identified. The exon size ranged from 59 to 657 nucleotides and exon-intron junctions followed known consensus sequences. Three of the five extracellular Ig-related domains (D1, D4 and D5) were confined to one exon each (E3, E5 and E6), whereas D2 and D3 were encoded by the same exon (E4). The latter exon corresponds to that involved in alternate splicing of rabbit SC. The membrane-spanning segment was confined to part of one exon (E8). The cytoplasmic tail was encoded by four exons (E8-E11), whose boundaries encompassed fairly well the structural determinants proposed to be responsible for intracellular sorting of SC in the rabbit. The polymorphic restriction site reported earlier for Pvu II was localized to the third intron.     

Cloning and characterization of the rRNA genes and flanking regions from Babesia bovis: use of the genes as strain discriminating probes

Three sets of rRNA genes (units 1S-3S) have been identified in Babesia bovis (Samford isolate). All three units are present in the same, probably single, copy number. The rRNA genes and flanking regions have been analysed by cloning, restriction mapping and DNA hybridization. The units are approximately 7 kb in length and have essentially identical restriction maps. In contrast the flanking regions exhibit significant restriction site differences. However, the regions upstream of all three units are related and sequences similar to part of the region upstream of units 1S and 3S are present in multiple copies in the genome. The downstream regions appear to be unrelated, but downstream from unit 1S is a region of at least 7 kb similar to a second region not closely linked to the rDNA units. The restriction enzyme site polymorphisms in the flanking regions of the equivalent units in different isolates allow ready discrimination among six different isolates of B. bovis.     

CpG Islands in Human X-Inactivation

Sequence comparison analyses have been carried out for 19 genes escaping X‐inactivation versus 73 genes subject to X‐inactivation, and 100 randomly chosen X chromosome genes versus 100 randomly chosen autosomal genes. The coding sequence of the genes and their upstream and downstream flanking sequences were investigated using a series of windows (1 kb, 2 kb, 5 kb, 10 kb and 100 kb). No significant difference in number of LINE‐L1 elements was observed in genes escaping X‐inactivation compared to genes subject to X‐inactivation. This result, therefore, does not support the suggestion that lack of LINE repeat elements is a key factor for genes escaping X‐inactivation. However, significantly reduced numbers of CpG islands and SINE MIR elements were found to be associated with genes escaping X‐inactivation. Compared to genes known to be inactivated, genes escaping X‐inactivation were observed to have fewer CpG islands, particularly within the 2 kb upstream flanking sequence close to the coding region. The results suggest that CpG islands may play a role in the process of X‐inactivation by providing sufficient DNA methylation targets for the maintenance of X‐inactivation. Lack of CpG islands may be a major reason for genes escaping X‐inactivation regulation.     

Cloning of a non-c-myc DNA fragment from the double minutes of a human colon carcinoid cell line

The cell line COLO 320 DM, derived from an untreated human colon carcinoid tumor, was subcloned to obtain a population (Cl 11) with an average of 37 double minutes (DM) per cell. Fractionation of the chromosomes by differential centrifugation yielded a fraction enriched in DM. DNA isolated from the DM-enriched fraction was inserted into the Pst I site of pBR322. One clone, p446, representative of a number of similar clones, contained a region complementary to genomic unique sequences (region p446U). Southern blot analysis using COLO 320 DNA, and DNA from two other cell lines derived from the same biopsy, COLO 320 HSR and COLO 321 HSR, demonstrated amplification and rearrangement of sequences complementary to p446U when compared with 28 different tumor and normal cell lines, some of which contained DM or homogeneously staining regions (HSR). COLO 320 DM Cl 11 had approximately 110 copies per cell of the p446U sequence, or three copies per DM. COLO 320 HSR, which contained one HSR, had 35 copies per cell, while COLO 321 HSR, which contained two HSR, had 700 copies. In addition, p446U did not hybridize with insert sequences of recombinant plasmid pHM(E + H), which includes the human c-myc coding region, 3 kb of upstream flanking sequences and 0.5 kb of downstream flanking sequences, or with an exon 3 probe, pMYC RI-CLA. Amplification of p446U was also not seen in cell lines containing amplified c-myc or N-myc genes. These results indicate that more than one sequence may be amplified in DM or HSR containing tumor cells, but that they need not be amplified together in other tumors.