Unusual sequences of two old, inactive human Alu repeats.

Two human Alu repeats terminating in an oligo(T) run rather than the usual A-rich 3' tail were isolated by library screening. Base sequence comparisons reveal that these unusual Alus are also exceptionally divergent from other Alu family members implying that they are evolutionarily old. Unlike other members of the family, they are not transcribed in vitro by RNA polymerase III (Pol III) suggesting a partial explanation for how Alu source genes might become inactive with age.     

Association of hY4 pseudogenes with Alu repeats and abundance of hY RNA-like sequences in the human genome.

Three loci having homology with the small human cytoplasmic RNA, hY4, were isolated from human genomic DNA libraries and sequenced. Each sequence contains dispersed mismatches as compared with hY4 RNA, is followed by an A-rich or A + T-rich sequence, and is bordered by direct repeats. Each of these loci, therefore, appears to constitute a small RNA class-III pseudogene. Surprisingly, two of the three loci are associated with Alu repeats. In the hY4.B7 locus, the hY4 sequence has integrated into the tail of an Alu element and in the hY4.F2 locus, an Alu sequence has inserted into the hY4 tail, confirming that A-rich tracts are preferential targets for retroposition. In addition, Southern blots with probes for each of the four hY RNAs indicate that hY RNA-like sequences are abundant in the human genome.     

Nucleotide sequences of mouse genomic loci including a gene or pseudogene for U6 (4.8S) nuclear RNA.

We have isolated four clones which hybridize with U6 (4.8S) nuclear RNA, a mammalian small nuclear RNA(nRNA), from DNA of BALB/C mouse liver. Their restriction maps are totally different from each other, indicating that they derived from different loci in the mouse genome. The nucleotide sequences around the hybridizing region in the three clones have been determined. One clone gives a gene that is co-linear with the U6 RNA. There is a sequence TATAAAT beginning 31 nucleotides upstream of the gene, which may suggest that the U6 RNA is transcribed by RNA polymerase II. The other two clones contain a pseudogene for the U6 RNA which has 7 or 9 nucleotide changes from the RNA. The pseudogenes are surrounded by radically different sequences from those surrounding the gene, and they are closely linked to a pseudogene for another snRNA, 4.5S-I RNA, or a part of highly repetitive an interspersed sequence B1.     

Cell free synthesis of some storage protein subunits by polyribosomes and RNA isolated from developing seeds of pea (Pisum sativum L.)

Polyribosomes which have template activity in the wheat germ system have been isolated from developing pea seeds. Some of the translation products have identical mobilities to the vicilin and legumin subunits by SDS-PAGE. Certain products were specifically immunoprecipitated with antisera prepared against purified vicilin and legumin fractions. Various RNA fractions including poly A-rich RNA have also been isolated from polyribosomes and shown to direct the synthesis of polyripeptides whose properties are similar to the storage protein subunits. The results are discussed in relationship to other investigations with seed storage protein biosynthesis in vitro.Abbreviations DTT dithiothreitol - SDS-PAGE SDS-polyacrylamide gel electrophoresis - TCA tricarboxylic acid     

Rna15 Interaction with the A-Rich Yeast Polyadenylation Signal Is an Essential Step in mRNA 3′-End Formation

In Saccharomyces cerevisiae, four factors [cleavage factor I (CF I), CF II, polyadenylation factor I (PF I), and poly(A) polymerase (PAP)] are required for maturation of the 3' end of the mRNA. CF I and CF II are required for cleavage; a complex of PAP and PF I, which includes CF II subunits, participates in polyadenylation, along with CF I. These factors are directed to the appropriate site on the mRNA by two sequences: one A-rich and one UA-rich. CF I contains five proteins, two of which, Rna15 and Hrp1, interact with the mRNA through RNA recognition motif-type RNA binding motifs. Previous work demonstrated that the UV cross-linking of purified Hrp1 to RNA required the UA-rich element, but the contact point of Rna15 was not known. We show here that Rna15 does not recognize a particular sequence in the absence of other proteins. However, in complex with Hrp1 and Rna14, Rna15 specifically interacts with the A-rich element. The Pcf11 and Clp1 subunits of CF I are not needed to position Rna15 at this site. This interaction is essential to the function of CF I. A mutant Rna15 with decreased affinity for RNA is defective for in vitro RNA processing and lethal in vivo, while an RNA with a mutation in the A-rich element is not processed in vitro and can no longer be UV cross-linked to the Rna15 subunit assembled into CF I. Thus, the recognition of the A-rich element depends on the tethering of Rna15 through an Rna14 bridge to Hrp1 bound to the UA-rich motif. These results illustrate that the yeast 3' end is defined and processed by a mechanism surprisingly different from that used by the mammalian system.     

In vitro selection of RNAs with increased tertiary structure stability.

An in vitro selection system was devised to select RNAs based on their tertiary structural stability, independent of RNA activity. Selection studies were conducted on the P4-P6 domain from the Tetrahymena thermophila group I intron, an autonomous self-folding unit that contains several important tertiary folding motifs including the tetraloop receptor and the A-rich bulge. Partially randomized P4-P6 molecules were selected based on their ability to fold into compact structures using native gel electrophoresis in the presence of decreasing concentrations of MgCl2. After 10 rounds of the selection process, a number of sequence alterations were identified that stabilized the P4-P6 RNA. One of these, a single base deletion of C209 within the P4 helix, significantly stabilized the P4-P6 molecule and would not have been identified by an activity-based selection because of its essential role for ribozyme function. Additionally, the sequence analysis provided evidence that stabilization of secondary structure may contribute to overall tertiary stability for RNAs. This system for probing RNA structure irrespective of RNA activity allows analysis of RNA structure/function relationships by identifying nucleotides or motifs important for folding and then comparing them with RNA sequences required for function.     

Structure and expression of the cAMP cell-surface receptor

Using antibodies specific for the 3',5'-cyclic AMP (cAMP) cell surface receptor of Dictyostelium discoideum, we have screened lambda gtll expression libraries and isolated a series of cDNAs derived from cAMP receptor mRNA during early development. The identity of the cDNA clones was verified by multiple criteria: 1) beta-galactosidase fusion proteins synthesized by isolated cDNA clones stain intensely with cAMP receptor directed antiserum, 2) these fusion proteins affinity purify antibodies specific for the cAMP receptor, 3) the cDNA probes hybridize to a 2 kb mRNA whose change in relative level of abundance during development parallels that of receptor mRNA as assayed by in vitro translation, 4) the 2 kb mRNA size equals that of receptor mRNA as determined by in vitro translation of size fractionated poly (A)+ RNA, and 5) RNA transcribed in vitro from cDNAs containing the entire protein-coding region produces a polypeptide by in vitro translation with an apparent molecular weight in close agreement with that of nascent cAMP receptor protein produced by in vitro translation of cellular RNA. The DNA sequence predicts an open reading frame of 392 amino acids. The deduced amino acid sequence contains seven domains enriched in hydrophobic residues. A model is proposed in which the cAMP cell-surface receptor traverses the lipid bilayer seven times in a pattern similar to that of other receptors, such as rhodopsin, which interact with G-proteins. The structural similarities suggest a gene family of related surface receptors from such evolutionarily diverse species as Dictyostelium, yeast, and mammals.     

The characterization of cDNA clones coding for wheat storage proteins.

Poly(A)+ RNA isolated from the developing wheat endosperm var. Chinese Spring, has been used as template for the construction of a cDNA library. Within the library, clones have been identified by in vitro translation of hybrid-selected mRNA which encode alpha/beta gliadin related sequences and gamma-gliadin related sequences. The DNA sequence of one such clone has been determined and it shows homology with that of a clone encoding a barley storage protein, B-hordein. The sequence includes a tandem DNA repeat which is discussed in relation to the generation of diversity within the gliadins.     

Comparison of RNA Polymerase Associated With Newcastle Disease Virus and a Temperature-Sensitive Mutant of Newcastle Disease Virus Isolated from Persistently Infected L Cells

An in vitro comparison was made of the RNA polymerase activity associated with Newcastle disease virus (NDVo) and three clones of the temperature-sensitive mutant (NDVpi) isolated from persistently infected L cells. Less polymerase activity was associated with the NDVpi clones. Also, compared to NDVo, an increase in incubation temperature from 32 to 37 or 42 C resulted in a marked decrease in polymerase activity for the temperature-sensitive mutants which coincided with their inability to replicate at 42 C.     

Human DNA sequences complementary to the small nuclear RNA U2.

Clones containing sequences complementary to the small nuclear RNA U2 were isolated from a human DNA library (1). Three clones, designated U2/4, U2/6 and U2/7 were purified and characterized by restriction enzyme cleavage, hybridization and heteroduplex analysis. Hybridization showed that the three clones each contained one single region which is complementary to U2 RNA. Restriction enzyme cleavage revealed furthermore that the inserted fragments in the three recombinants are different. Heteroduplex analysis identified a 240-380 bp long duplex region in each heteroduplex which includes sequences complementary to U2 RNA. Heteroduplexes between clones U2/4 and U2/7 as well as between U2/4 and U2/6 revealed two additional approximately 200 bp long homologies. The remainder of the inserts were found to lack measurable sequence homology. Two fragments from clone U2/4 were subcloned in the pBR322 vector and the subclones were used to determine the nucleotide sequence of a region in clone U2/4 which is complementary to U2 RNA. A comparison between the established sequence and the sequence for rat U2 RNA (2) reveals several discrepancies.     

Comparison of RNA polymerase associated with Newcastle disease virus and a temperature-sensitive mutant of Newcastle disease virus isolated from persistently infected L cells.

An in vitro comparison was made of the RNA polymerase activity associated with Newcastle disease virus (NDVo) and three clones of the temperature-sensitive mutant (NDVpi) isolated from persistently infected L cells. Less polymerase activity was associated with the NDVpi clones. Also, compared to NDVo, an increase in incubation temperature from 32 to 37 or 42 C resulted in a marked decrease in polymerase activity for the temperature-sensitive mutants which coincided with their inability to replicate at 42 C.