U4 small nuclear RNA pseudogenes from rat genome have common truncated 3'-ends

Four U4 RNA pseudogenes were isolated and characterized from a rat genomic bank. The four pseudogenes contained sequences completely homologous to U4 RNA from nucleotides 1 to 67 and had common truncated 3'-ends. Three of the four pseudogenes were flanked by 14 to 18 nucleotide-long direct repeats. The structural features of these four U4 RNA pseudogenes are consistent with the hypothesis that these pseudogenes arose by RNA self-primed complementary DNA synthesis and integration into the genome (Van Arsdell et al., Cell 26:11-17, 1981).     

Isolation and characterization of a human U3 small nucleolar RNA gene

U3 RNA is an abundant, capped, small nucleolar RNA, implicated in the processing of preribosomal RNA. In this study, a DNA clone coding for U3 RNA (clone U3-1) was isolated from a human genomic library and characterized. The DNA sequence was identical to that of human U3 RNA isolated from HeLa cells. The flanking regions showed homology to the enhancer, promoter, and 3'-processing signal found in U1 and U2 snRNA genes. Further, the recently identified "U3 box" (GATTGGCTGCN10TATGTTAATTATGG) of rat U3 genes (Stroke and Weiner, (1985) J. Mol. Biol. 184, 183-193), was also found in the human U3 gene. This gene was transcribed in Xenopus oocytes; it is the first cloned true human U3 gene.     

Isolation of an active gene and of two pseudogenes for mouse U7 small nuclear RNA

Three U7 RNA-related sequences were isolated from mouse genomic DNA libraries. Only one of the sequences completely matches the published mouse U7 RNA sequence, whereas the other two apparently represent pseudogenes. The matching sequence represents a functional gene, as it is expressed after microinjection into Xenopus laevis oocytes. Sequence variations of the conserved cis-acting 5' and 3' elements of U RNA genes may partly explain the low abundance of U7 RNA.     

Nonrandom integration of human U4 RNA pseudogenes.

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.     

Isolation and characterization of Drosophila melanogaster U2 small nuclear RNA genes

We describe here the organization of DNA sequences complementary to Drosophila melanogaster U2 small nuclear (sn) RNA. From a genomic library we isolated two recombinants containing two genes each. Genomic reconstruction experiments and Southern analysis revealed that D. melanogaster possesses only four to five U2 snRNA genes or very closely related sequences. The nucleotide sequence of one of the clones analysed shows 77% homology with rat U2 snRNA. A stretch of 12 nucleotides that has been implicated in heterogeneous nuclear RNA splicing is conserved between rat and Drosophila. The genomic organization of these genes is very similar in different melanogaster strains but diverges highly in different Drosophila species.     

Nucleotide sequence and organization of full length human U4 RNA pseudogenes

Two loci encoding human U4 RNA, designated U4/7 and U4/14, have been isolated and sequenced. Both are pseudogenes in that their sequences do not match any identified human U4 RNA species perfectly. The U4/7 locus harbours a full-length pseudogene of 144 bp with eight base substitutions in the structural region. This pseudogene might be derived from a hitherto unidentified human U4 RNA gene. The second locus, U4/14, has a complex structure; the structural sequence of a U4 gene has apparently been integrated into an Alu sequence.     

Structures of four human pseudogenes for U7 small nuclear RNA

Four U7 RNA-related sequences were isolated from a human genomic DNA library. None of the sequences completely match the published human U7 RNA sequence and all of them contain features typical of reverse-transcribed pseudogenes.     

Identification and characterization of three genes and two pseudogenes on chromosome 13

A study was conducted on the feasibility of isolating genes and pseudogenes that map to chromosome 13 by a hybridization-based approach using a 13-specific library and pools of repeat-free cDNA clones. Five pairs of cDNA and chromosome 13 genomic clones were identified and characterized. Partial or full-length sequence was derived from all cDNAs, and database searches were performed for putative gene identification. Partial sequence was also obtained from the chromosome 13 genomic clones for comparison with those of the hybridizing cDNAs. As a result of these analyses we identified three genes, a putative homologue of a porcine mRNA encoding an unidentified hepatic protein, a putative homologue of a yeast integral membrane protein, and a gene for a translationally controlled tumor protein, and two processed pseudogenes, ribosomal proteins L23a and S3a. The latter was formerly identified as the v-fos transformation effector gene, Fte-1, and recently cited as a possible candidate for the BRCA2 gene on chromosome 13. All genes and pseudogenes were localized to cytogenetic bands by in situ hybridization of metaphase chromosomes with probes derived from the chromosome 13 genomic clones.     

Isolation and characterization of two linked mouse U1b small nuclear RNA genes.

A 6.9 kilobase Eco R1 fragment containing genes for two U1 RNAs has been isolated from a library of mouse DNA. The two genes code for an RNA which is very similar, if not identical, to mouse U1b RNA as judged by S1 nuclease mapping. This RNA is one base longer than the mouse U1a RNA, human U1 RNA, and rat U1 RNA and differs in six nucleotide substitutions from rat U1 RNA. The two genes are five kilobases apart and the U1 RNAs are coded for on opposite strands of the DNA with the 5' ends juxtaposed. The sequences flanking the genes are identical for 700 bases 5' to the gene and at least 80 bases 3' to the gene.     

Isolation and characterization of three different forms of arylamidase from rat skeletal muscle

An arylamidase hydrolysing L-leucine-4-nitroanilide was extracted from rat skeletal muscle homogenate and furified by means of anion-exchange chromatography on DEAE-Sephadex A-50 followed by gel filtration on Sephadex G-150 and Sepharose 6B. The enzyme was isolated in the form of three different protein complexes that differ in molecular weight, kinetic data, and sensitivity to metal ions. As studied by SDS-gel electrophoresis and repeated gel chromatography on Sepharose 6B these forms are: 1. a stable monomer (A1) of M_r 122 000; 2. a stable dimer (A2) of M_r 244 000; and 3. a stable polymer (A3) of more than M_r 4·10⁶. The arylamidase was optimally active at pH 7.3 and did not require metal ions. Treatment with 1,10-phenanthroline resulted in complete inactivation, the activity could be restored by the addition of manganous chloride. The sulphhydryl-blocking reagent 4-hydroxymercuribenzoate strongly inactivated the arylamidase, this inhibition could be reversed by the addition of 2-mercaptoethanol. Addition of phenylmethylsulfonyl fluoride had no effect on the enzyme activity. Furthermore, the influence of metal ions as well as the substrate specificity were investigated and compared for all three forms of arylamidase.     

Isolation and characterization of fluorescence-enhancing RNA tags

Methods for the visualization of RNAs are urgently needed for studying a wide variety of cellular processes. Here we report on-bead screening of RNA libraries and its application to the isolation of specific fluorescence-enhancing RNA sequences. A one-bead-one-compound combinatorial RNA library with over one million different sequences was synthesized using the split-and-mix method. Solid-phase synthesis of 30 mer RNAs was performed on 15 ??m and 60 ??m diameter polystyrene beads bearing a non-cleavable linker. The RNA-derivatized beads were incubated with the well-established FlAsH pre-fluorophore and then screened for fluorescence enhancement, either by manually picking the brightest beads under a fluorescence microscope or by sorting with a FACS instrument. A protocol was established for sequence determination from single beads. While numerous RNA sequences showed increased fluorescence when immobilized, only few of them influenced the fluorescence properties of the FlAsH dye when detached from the beads. One of these sequences was found to induce a bathochromic shift in the excitation (from 492 to 510 nm) and emission (from 512 to 523 nm) maxima. This shift was accompanied by a 3.6-fold fluorescence enhancement of FlAsH fluorescence intensity. Mutation studies on the sequence revealed a rather robust structural motif.     

Human U1 RNA pseudogenes may be generated by both DNA- and RNA-mediated mechanisms.

Analysis of cloned human genomic loci homologous to the small nuclear RNA U1 established that such sequences are abundant and dispersed in the human genome and that only a fraction represent bona fide genes. The majority of genomic loci bear defective gene copies, or pseudogenes, which contain scattered base mismatches and in some cases lack the sequence corresponding to the 3' end of U1 RNA. Although all of the U1 genes examined to date are flanked by essentially identical sequences and therefore appear to comprise a single multigene family, we present evidence for the existence of at least three structurally distinct classes of U1 pseudogenes. Class I pseudogenes had considerable flanking sequence homology with the U1 gene family and were probably derived from it by a DNA-mediated event such as gene duplication. In contrast, the U1 sequence in class II and III U1 pseudogenes was flanked by single-copy genomic sequences completely unrelated to those flanking the U1 gene family; in addition, short direct repeats flanked the class III but not the class II pseudogenes. We therefore propose that both class II and III U1 pseudogenes were generated by an RNA-mediated mechanism involving the insertion of U1 sequence information into a new chromosomal locus. We also noted that two other types of repetitive DNA sequences in eucaryotes, the Alu family in vertebrates and the ribosomal DNA insertions in Drosophila, bore a striking structural resemblance to the classes of U1 pseudogenes described here and may have been created by an RNA-mediated insertion event.     

Isolation and characterization of three unusual membrane glycopeptides present in rat intestinal endoplasmic reticula

Three glycopeptides have been isolated from the mucosal homogenates of the rat small intestine without using proteolysis. These glycopeptides appear to be localized exclusively in the membranes of the endoplasmic reticula. Although they have similar molecular weights of about 2550 and have similar amino acid compositions, they differ in the carbohydrate constituents. The major glycopeptide has 2 mol glucose per polypeptide chain while the two other glycopeptides contain 1 mol fucose, mannose and galactose with either 1 or 2 mol glucose. No hexosamine or sialic acid was detected in any of the glycopeptides. An unusual physical property was found associated with these glycopeptides. Below pH 6.5 they formed a precipitate which prevented them from diffusing through a dialysis membrane and allowed them to be rapidly purified following solubilization from the membrane. These glycopeptides appear to represent a new group of heretofore uncharacterized membrane constituents which may play a role in some function specific for the endoplasmic reticula.     

Genes and pseudogenes for human U2 RNA: Implications for the mechanism of pseudogene formation

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 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.     

Isolation and characterization of oat globulin messenger RNA

When polyadenylated RNA, isolated from membrane-bound polysomes extracted from developing oat (Avena sativa L.) seeds, was translated in vitro in the rabbit reticulocyte system, two polypeptides of about 58 and 60 kilodaltons were immunoprecipitated by anti-oat globulin antibody. No electrophoretic bands corresponding to the 40 and 20 kilodalton polypeptides of oat globulin were present. However, when in vivo labeled extracts were immunoprecipitated with anti-oat globulin antibody, three groups of polypeptides (60, 40, and 20 kilodaltons) were present. It therefore seems probable that the two large polypeptides (58 and 60 kilodaltons) were precursors of the 40 and 20 kilodalton polypeptides. When the polyadenylated RNA coding for these polypeptides was size fractionated on a sucrose density gradient, it sedimented near the 18S region of the gradient. Translation of the RNA from the gradient fractions and immunoprecipitation of translation products indicated that the template for the 58 to 60 kilodalton `putative' precursors of oat globulin was probably the RNA which was approximately 18S in size.