Showing papers by "Carl W. Schmid published in 1993"

Developmental differences in methylation of human Alu repeats.

Abstract: Alu repeats are especially rich in CpG dinucleotides, the principal target sites for DNA methylation in eukaryotes. The methylation state of Alus in different human tissues is investigated by simple, direct genomic blot analysis exploiting recent theoretical and practical advances concerning Alu sequence evolution. Whereas Alus are almost completely methylated in somatic tissues such as spleen, they are hypomethylated in the male germ line and tissues which depend on the differential expression of the paternal genome complement for development. In particular, we have identified a subset enriched in young Alus whose CpGs appear to be almost completely unmethylated in sperm DNA. The existence of this subset potentially explains the conservation of CpG dinucleotides in active Alu source genes. These profound, sequence-specific developmental changes in the methylation state of Alu repeats suggest a function for Alu sequences at the DNA level, such as a role in genomic imprinting.

Proposed roles for DNA methylation in Alu transcriptional repression and mutational inactivation

Abstract: Methylation at CpG dinucleotides to produce 5 methyl cytosine (5me-C) has been proposed to regulate the transcriptional expression of human Alu repeats. Similarly, methylation has been proposed to indirectly favor the transpositional activity of young Alu repeats by transcriptionally inactivating older Alu's through the very rapid transition of 5me-C to T. Both hypotheses are examined here by RNA polymerase III (Pol III) in vitro transcription of Alu templates using HeLa cell extracts. A limiting factor represses the template activity of methylated Alu repeats. Competition by methylated prokaryotic vector DNA's relieves repression, showing that the factor is not sequence specific. This competitor has no effect on the activity of unmethylated templates showing that the repressor is highly specific toward methylated DNA. While methylation of a single pair of CpG dinucleotides in the A box of the Poll III promoter is sufficient to cause repression, methylation elsewhere within the template also causes repression. The repressor causing these effects on the Pol III directed transcription of Alu repeats is thought to be a previously reported, repressor for Pol II directed templates. Young Alu repeats are transcriptionally more active templates than a representative older Alu subfamily member. Also, younger Alu's form stable transcriptional complexes faster, potentially giving them an additional advantage. The mutation of three CpG's to CpA's within and near the A box drastically decreases both the template activity and rate of stable complex formation by a young Alu member. The sensitivity of Alu template activity to CpG transitions within the A box partially explains the selective transpositional advantage enjoyed by young Alu members.

Phylogenetic isolation of a human alu flounder gene: Drift to new subfamily identity

Abstract: A severe bottleneck in the size of the PV Alu subfamily in the common ancestor of human and gorilla has been used to isolate an Alu source gene. The human PV Alu subfamily consists of about one thousand members which are absent in gorilla and chimpanzee DNA. Exhaustive library screening shows that there are as few as two PV Alus in the gorilla genome. One is gorilla-specific, i.e., absent in the orthologous loci in both human and chimpanzee, suggesting the independent retrotranspositional activity of the PV subfamily in the gorilla lineage. The second of these two gorilla PV Alus is present in both human and chimpanzee DNAs and is the single PV Alu known to precede the radiation of these three species. The orthologous Alu in gibbon DNA resembles the next older Alu subfamily. Thus, this Alu locus is originally templated by a non-PV source gene and acquired characteristic PV sequence variants by mutational drift in situ, consequently becoming the first member and presumptive founder of this PV subfamily.

Evolutionary analyses of repetitive DNA sequences.

Abstract: Publisher Summary This chapter describes the evolutionary analyses of repetitive DNA sequences. The study of repetitive DNA sequences is refined by advances in cloning, sequencing, and oligonucleotide synthesis. Whereas the original studies of repeated DNA evolution analyzes whole families of repeated DNA sequences using genomic hybridization techniques, it is now possible to use cloning and DNA sequence analysis to define subfamilies of repeated DNA sequences. These subfamilies may then be characterized rapidly utilizing other approaches, such as specific oligonucleotide probes and the polymerase chain reaction (PCR). It is difficult to improve on direct DNA sequence comparisons for evolution studies of the repeated DNA sequences. The only drawback is that these studies are relatively labor intensive, limiting the experimental sample to a much smaller one than can be studied using hybridization procedures. The chapter highlights the special considerations for repetitive DNA evolution.

Mobility of short interspersed repeats within the chimpanzee lineage

Abstract: The PV subfamily of Alu repeats in human DNA is largely composed of recently inserted members. Here we document additional members of the PV subfamily that are found in chimpanzee but not in the orthologous loci of human and gorilla, confirming the relatively recent and independent expansion of this Alu subfamily in the chimpanzee lineage. As further evidence for the youth of this Alu subfamily, one PV Alu repeat is specific to Pan troglodytes, whereas others are present in Pan paniscus as well. The A-rich tails of these Alu repeats have different lengths in Pan paniscus and Pan troglodytes. The dimorphisms caused by the presence and absence of PV Alu repeats and the length polymorphisms attributed to their A-rich tails should provide valuable genetic markers for molecular-based studies of chimpanzee relationships. The existence of lineage-specific Alu repeats is a major sequence difference between human and chimpanzee DNAs.