Biomedical subjects
Publications and source records attributed to K Hammarström.
The gene cluster for human U2 RNA has been mapped to chromosome 17q21 by in situ hybridization and hybridization analysis of DNA from mouse/human somatic cell hybrids.
Four loci for human U4 RNA have been characterized by DNA sequence analysis. The results show that all four loci represent pseudogenes, which are flanked by direct repeats. Three of the pseudogenes, designated U4/5, U4/6, and U4/8, have very similar structures; they are all truncated and contain the first 67 to 68 nucleotides of the U4 RNA sequence. Their properties suggest that they were created by integration of truncated cDNA copies of the U4 RNA into new chromosomal sites. An interesting observation was that their flanking regions exhibit sequence homology. A purine-rich 5'-flanking sequence 12 to 13 nucleotides long is almost perfectly conserved in all three loci. Boxes of homology were also found on the 3' side when the U4/6 and U4/8 loci were compared. The U4/4 locus has a slightly different structure; the pseudogene matches the first 79 nucleotides of U4 RNA, but contains a greater number of mutations than the other pseudogenes. Taken together, the results suggest that a frequently occurring type of pseudogene for human U4 was created by a RNA-mediated mechanism and that the integration sites have features in common.
Three loci, designated U2/4, U2/6 and U2/7, which contain sequences related to human U2 RNA, have been studied. The U2/6 locus contains a tandem array of bona fide U2 genes. U2/4 and U2/7, in contrast, contain pseudogenes of whose sequences deviate significantly from that of mammalian U2 RNA. The two pseudogenes appear to have been created by different mechanisms. The sequences that flank the pseudogene in the U2/4 locus lack homology to the corresponding sequences in functional human U2 genes, except for 10 base-pairs immediately following the 3' end. The conserved 3'-flanking segment is homologous to those nucleotides that are present in a U2 RNA precursor. No direct repeats flank the pseudogene in the U2/4 locus. The observations thus suggest that a complementary DNA copy of the U2 RNA precursor was inserted into a blunt-ended chromosomal break to generate the U2/4 locus. The U2/7 locus, in contrast, revealed flanking sequence homology when compared to functional U2 genes, both on the 5' and 3' sides of the pseudogene. The homology was interrupted on both sides by repetitive sequences belonging to the Alu family. On the 5' side the homology continues beyond the Alu repeats whereas on the 3' side it ends precisely at the Alu repeat. This Alu repeat is inserted in a region where a homocopolymeric region of alternating C and T residues is located in functional U2 loci. The observed organization of the U2/7 locus suggests that a previously functional U2 locus was invaded by Alu repeats and subsequently accumulated base substitutions to become a pseudogene.
The use of displacement electrophoresis (synonymous to isotachophoresis, steady-state stacking, and moving boundary electrophoresis) for recovery of DNA fragments from agarose and polyacrylamide gels is described. Complete recovery of DNA molecules ranging from oligonucleotides to 20 000-basepairs-long fragments was achieved. The DNA is recovered in a small volume (0.1-0.3 ml) and can be used directly in enzyme-mediated cleavage and ligation reactions. The recovered DNA contained no inhibitory contaminants as revealed by ligation or restriction enzyme cleavage.
Genes for the human small nuclear RNA U2 are present within 6.2-kilobase-pair-long tandem repeats. The haploid human genome contains approximately 20 such repeats, organized in one or a few very large clusters.
Three clones U1-1, U1-6, and U1-8 containing sequences related to human U1 RNA have been studied by sequence analysis. The results show that each of the three clones represents a distinct locus. The U1-6 locus is closely related to the HU1-1 locus, which is believed to represent a functional U1 gene. The U1-1 and U1-8 loci are pseudogenes by definition, since they contain sequences that are closely related to but not identical with the human U1 RNA sequence. The U1-6 locus contains the sequence T-A-T-A-T close to the 5'-end of the U1 sequence but it is unclear if this represents the promoter. When the U1-8 locus was compared to the U1-6 locus, it was observed that the 5'-flanking sequences, except in the immediate vicinity of the pseudogene, are as well-conserved as the U1-related sequence itself, at least up to position -220. The high degree of homology in the 5'-flanking region suggests that U1 genes have a much more strict sequence requirement with regard to 5'-flanking sequences than most other eukaryotic genes. The U1-6 and U1-8 loci contain the sequence T-A-T-G-T-A-G-A-T-G-A between positions -211 and -221. An identical sequence is present in the equivalent position in the HU1-1 locus, and may represent the promoter. The high degree of conservation in the postulated promoter region indicates that pseudogenes like U1-8 possibly could be expressed. A truncated U1-related sequence is present between 106 to 150 nucleotides upstream from the U1 gene/pseudogene in the U1-6, the U1-8 and the HU1-1 loci, suggesting that the U1 genes may have been clustered early in evolution. The U1-1 locus has a strikingly different structure from the U1-8 locus; the pseudogene itself is as closely related to the U1 RNA sequence as is the U1-8 pseudogene but the flanking sequences, both on the 5' and the 3' side, share no detectable homology with the corresponding regions in the U1-6 or U1-8 loci. It may therefore be postulated that small nuclear RNA pseudogenes are created by several different mechanisms.
The human DNA library of Lawn et al. (1978) was screened for sequences complementary to the small nuclear (sn) RNA U4. Several positive clones were identified by screening 100 000 recombinants, indicating that U4 sequences like other snRNA sequences are dispersed in the human genome. One recombinant was characterized in detail by subcloning a Bg/II fragment 1.9 kilobases (kb) long in the pBR322 plasmid. The subcloned fragment was partially sequenced and the results revealed a pseudogene for U4 RNA. The pseudogene was found to have a remarkable structure; it contains a sequence that, except in two positions, matches the first 68 nucleotides of the human U4 RNA sequence and the pseudogene is, moreover, flanked by perfect direct repeats 20 bp long. The results support the model of van Arsdell et al. (1981) suggesting that certain snRNA pseudogenes arise by reverse transcription of the RNA followed by integration of the cDNA copy at a new chromosomal locus.
Pancreatic islets were microdissected from ob/ob mice, loaded for 2 h with 45Ca and perfused with calcium-deficient medium. Irrespective of the glucose and calcium concentrations in the loading medium, increased glucose in the perfusion medium resulted in reduced amounts of radioactivity in the perfusate. A glucose inhibition of 45Ca washout was also evident when the specific radioactivity of the islets approached that of the labeling medium, indicating that the effect was not simply due to isotopic dilution. The depression of 45Ca washout diminished after culture of the islets in a serum-free medium and it was absent in islets taken from mice homozygous for the gene diabetes. The glucose effect became less pronounced when 50 micron D-600, an inhibitor of the calcium inward transport, was added to the calcium-deficient perfusion medium and abolished in the presence of 20 mM Ca-EGTA. The inhibition of the 45Ca washout observed is not necessarily due to a direct glucose interaction with the outward calcium transport but may also result from stimulation of the uptake and intracellular trapping of the cation.