Maxine F. Singer

Highly Repeated Sequences in Mammalian Genomes

Publisher Summary This chapter discusses the structure and organization of mammalian, highly repeated sequences at the molecular level. There is a description of tandemly repeated sequences, that are, satellites, and the segments that are interspersed among other genomic DNA sequences. The methods for the analysis of repeated DNA sequences are measurement of DNA renaturation kinetics and isopycnic centrifugation in gradients of CsCl and CsSO4. Eukaryote genomes can be divided into classes of DNA sequences according to the reiteration frequency. Many highly repeated sequences are in long tandem arrays. Some repetitive sequences are dispersed throughout major portions of genomes amid either other repeated sequences or sequences present only once per genome, that are, uniquesequences. The characteristic organizational feature of satellites and cryptic satellites is the tandem repetition of a unit DNA sequence. Satellite arrays resist separation by isopycnic centrifugation and instead remain within the main density fraction of genomic DNA. The repeat unit and its tandem organization can be revealed by restriction endonuclease digestion.

Deficient activity of guanine nucleotide regulatory protein in erythrocytes from patients with pseudohypoparathyroidism

Abstract The activity in two assay systems of guanine nucleotide regulatory protein was significantly lower in red cells from pseudohypoparathyroid patients compared to that of normal subjects. The results support the hypothesis that deficient activity of the guanine nucleotide regulatory protein is the molecular basis for hormone resistance in this inherited disorder.

Sequence-specific single-strand RNA binding protein encoded by the human LINE-1 retrotransposon

Previous experiments using human teratocarcinoma cells indicated that p40, the protein encoded by the first open reading frame (ORF) of the human LINE‐1 (L1Hs) retrotransposon, occurs in a large cytoplasmic ribonucleoprotein complex in direct association with L1Hs RNA(s), the p40 RNP complex. We have now investigated the interaction between partially purified p40 and L1Hs RNA in vitro using an RNA binding assay dependent on co‐immunoprecipitation of p40 and bound RNA. These experiments identified two p40 binding sites on the full‐length sense strand L1Hs RNA. Both sites are in the second ORF of the 6000 nt RNA: site A between residues 1999 and 2039 and site B between residues 4839 and 4875. The two RNA segments share homologous regions. Experiments involving UV cross‐linking followed by immunoprecipitation indicate that p40 in the in vitro complex is directly associated with L1Hs RNA, as it is in the p40 RNP complex found in teratocarcinoma cells. Binding and competition experiments demonstrate that p40 binds to single‐stranded RNA containing a p40 binding site, but not to single‐stranded or double‐stranded DNA, double‐stranded RNA or a DNA–RNA hybrid containing a binding site sequence. Thus, p40 appears to be a sequence‐specific, single‐strand RNA binding protein.

Helix formation between polyribonucleotides and purines, purine nucleosides and nucleotides. II

A number of monomeric nucleic acid components react with polynucleotides to form well-defined complexes having helical structures similar to those formed between corresponding pairs of high polymers. The monomer can be a purine or a purine nucleoside or nucleotide, but no pyrimidine derivative has yet been found to form such complexes. The phenomenon has been investigated by infrared spectroscopy and optical rotation and by use of an enzyme of known sensitivity to secondary structure. Base-pairing specificity of interaction is observed, and the components react in definite stoichiometric ratios. The dependence of the stabilities of some of the complexes upon concentration of reactants, concentration of salt, and upon temperature has been determined.

Making sense out of LINES: long interspersed repeat sequences in mammalian genomes

Abstract The highly repeated DNA sequences that are dispersed in mammalian genomes have seemed hopelessly complex. Because of the high copy-numbers of family members (10 4 to more than 10 5 ) and the absence of any clues about their function, they were relegated to the junk DNA category. Many investigators consider them simply as boring road-blocks to the mapping of genomes. Recent experiments, however, suggest that at least some of these sequences are functional and, as a result, questions about them can be more carefully framed.

Defining the beginning and end of KpnI family segments.

Comparison of the sequences at the ends of several newly cloned and full length members of the monkey KpnI family with one another and with previously described monkey and human segments defines the nucleotide sequence at the two termini. No terminal repeats either direct or inverted are noted within full length family members which may or may not be immediately flanked by direct repeats. At the 3′ terminus, several family members have polyadenylation signals followed by a d(A)‐rich stretch. The genomic frequency of segments within the full length element increases markedly from the 5′ to the 3′ terminus, consistent with the cloning of various truncated family members. One such truncated version joined to a low copy number DNA segment is inserted in monkey alpha‐satellite where the combination appears to have been amplified in conjunction with the satellite itself.

Organization of African green monkey DNA at junctions between α-satellite and other DNA sequences

Segments of African green monkey DNA containing sequences of the highly reiterated cryptic satellite DNA called α-satellite were selected from a library in λ bacteriophage. This λ library was constructed to enrich for monkey segments that contain (1) irregular regions of α-satellite and (2) α-satellite linked to other monkey sequences. At least 11 of 15 cloned monkey segments between 13 × 103 and 16 × 103 base-pairs in length, selected by hybridization to α-satellite, also include other monkey sequences. In general, α-satellite sequences close to the junctions with non-α-satellite DNA contain an abundance of divergent forms compared to the average frequency of such forms within total α-satellite. Many of the cloned segments are missing some of the HinIII sites that occur once in most monomer units of α-satellite, and likewise several of the cloned segments contain restriction sites that rarely occur in α-satellite as a whole. In some segments HinIII sites occur that are spaced at distances other than the basic multiple of 172 base-pairs. At least one of the cloned segments, however, is composed mainly of typical 172 base-pair long α-satellite monomer units. Several of these cloned DNAs have been mapped by restriction endonuclease digestion and Southern blot analysis and the arrangements of α-satellite and non-α-satellite sequences have been determined. In addition to segments that contain a boundary where satellite meets other types of sequence, some contain two such boundaries and thus satellite flanks a non-α-satellite segment. Further, two different types of non-α-satellite sequence appear to be common to more than one phage, perhaps indicating some recurring organization at boundaries.

Sequence relationships between single repeat units of highly reiterated African Green monkey DNA

Individual monomer and dimer units of the highly repeated alpha-component DNA of African Green monkeys were isolated and amplified by molecular cloning in pBR322. The purified sequences were characterized by digestion with restriction endonucleases and by primary nucleotide sequence analysis. Comparison of the cloned units with the 172 base pair long sequence representing the most abundant nucleotide at each position in the set of sequences comprising alpha-component allows the following conclusions. The set of sequences comprising alpha-component is made up of a very large number of related but slightly divergent sequences. Two neighboring repeats of the monomer unit are not necessarily more similar to one another than are randomly isolated monomers.

Degradation of polyuridylic acid by ribonuclease II: Protection by ribosomes

Abstract It has been suggested that both polynucleotide phosphorylase and the potassium-activated phosphodiesterase (RNase II) are involved in the degradation of messenger RNA in Escherichia coli . If this suggestion holds, it follows that a control mechanism which protects messenger RNA from degradation long enough for it to be translated into protein must also exist. One common proposal is that ribosomes may protect messenger RNA from degradation, and direct evidence for this notion is provided in this report. Polyuridylic acid is used as a model for messenger RNA. The experiments indicate that when polyuridylic acid is bound to the ribosomes in such a fashion that it does not readily dissociate, protection from degradation is observed. The requisite stability of the polyuridylic acid-ribosome complex can be achieved by low temperature (10 °C), by treating the complex with formaldehyde, or by the addition of transfer RNA to give a transfer RNA-poly U-ribosome complex. The experiments reported here indicate that RNase II and polynucleotide phosphorylase degrade poly U chains from the 3′-hydroxyl end of the chain to the point of ribosome attachment; the remainder of the polyribonucleotide is protected. Using these data along with current theories and knowledge on transcription and translation, a model for the control of messenger RNA degradation in E. coli is proposed.

Interruption of an alpha-satellite array by a short member of the KpnI family of interspersed, highly repeated monkey DNA sequences.

We describe here the interruption of a cloned African green monkey alpha-satellite array by an 829-base-pair-long nonsatellite DNA segment. Hybridization experiments indicate that the sequences within the interruption are homologous to segments frequently found in the 6-kilobase-pair-long members of the KpnI family of long, interspersed repeats. These data confirm and extend earlier results suggesting that sequences common to the KpnI family can occur independently of one another and in segments of variable lengths. The 829-base-pair-long segment, which is termed KpnI-RET, contains a terminal stretch of adenosine residues preceded by two typical but overlapping polyadenylation sites. KpnI-RET is flanked by direct repeats of a 14-base-pair-long segment of alpha-satellite that occurs only once in the satellite consensus sequence. These structural features suggest that KpnI-RET was inserted into the satellite array as a movable element.