Identification and characterization of new human medium reiteration frequency repeats.

We report nine new families of human medium reiteration frequency interspersed repetitive elements (MER elements). They were identified by computer-assisted analyses. Six of them were independently confirmed as repetitive families by DNA-DNA hybridization, and the number of elements for each of these families was estimated by plaque hybridization assay. The involvement of some of the reported MER elements in genetic rearrangements is demonstrated.

Medium reiteration frequency repetitive sequences in the human genome.

Fourteen novel medium reiteration frequency (MER) families were found, in the human genome, by using two different methods. Repetition frequencies per haploid human genome were estimated for each of these families as well as for six previously described MER DNA families. By these measurements, the families were found to contain variable numbers of elements, ranging from 200 to 10,000 copies per haploid human genome.

Affinity chromatography of a sequence-specific DNA binding protein using Teflon-linked oligonucleotides.

An affinity matrix was constructed by synthesis of a DNA oligonucleotide on a Teflon fiber support followed by deblocking and hybridization of the complementary strand. It was used to purify a sequence-specific binding protein at least 100-fold to near homogeneity. This matrix is easily fabricated on an automated DNA synthesizer, contains high levels of attached DNA, and has superior mechanical properties. It should be generally useful for affinity chromatography of DNA binding proteins.

A composite transposon 3' to the cow fetal globin gene binds a sequence specific factor.

Two unusual sequence organizations were found within the beta-globin locus of the cow. Each was a composite, consisting of closely linked Alu-type repeats with a short stretch of genomic non-repetitive sequence, called a lagan, sandwiched between. One lagan was found 3' to the fetal globin gene, while the second lay between the adult globin gene and a globin pseudogene. Southern blot analysis indicated that both lagans appeared twice within the cow haploid genome, with the second copies lying outside the cow beta-globin locus. One of these non-globin locus homologues was cloned and subjected to sequence analysis. Comparison of the DNA sequence data showed that the lagan-Alu composite was transposed as a unit. The lagan 3' to the cow fetal globin gene contains the recognition site for a sequence specific DNA binding factor. This factor was present in extracts from fetal, but not from adult cow tissues.

Dual evolutionary modes in the bovine globin locus.

Five bovine globin pseudogenes were subjected to sequence analysis. These genes include the three pseudogenes in the beta-type globin gene cluster as well as two allelic forms. Comparison of the sequences with those of the adult and fetal bovine globin genes shows that together they form a multigene family that was created by large-scale duplication. The structures are explained by invoking sequence exchange mediated by gene conversion. After their creation these genes evolved in a concerted fashion, exchanging sequence freely by intrachromosomal gene conversion. Subsequently, one by one, the genes were uncoupled from this exchange. This was accomplished by the creation of nonhomologies that formed barriers to gene conversion. These nonhomologies were several hundred bases in length and were formed by either deletion or by insertion of short repetitive sequences within the gene structures. In this way the genes made the transition from a rapid, coupled mode to a slow, solitary mode of evolution. Allelic gene polymorphisms were distributed inhomogeneously in the bovine globin family. It is proposed that this was due to interruption of interchromosomal gene conversion by a recent pseudogene duplication in the fetal globin gene cluster.

Concerted evolution of the cow epsilon 2 and epsilon 4 beta-globin genes.

The nucleotide sequences of the cow epsilon 2 and epsilon 4 globin genes were determined. The sequences were 95% identical. These genes arose via a four-gene block duplication that also gave rise to the bovine fetal (gamma) and adult (beta) genes. Their deduced amino acid sequences are unlike any previously reported fetal or adult globins; rather, comparison to other mammalian globin genes indicates that they are embryonic in nature. The sequence data indicate that these two genes have converted each other during evolution. Pairwise comparison to the corresponding goat genes shows greater similarity between paralogues than between more directly related orthologues. This is in direct contrast to the situation between the cow and goat fetal and adult genes. These observations suggest that the frequency of DNA conversion or the fixation of conversion events may vary in different locations of the cow beta-globin cluster.

Structure and organization of the bovine beta-globin genes.

Genomic clones spanning the entire cow beta-globin gene locus have been isolated and characterized. These clones demonstrate that the linkage of embryonic-like (epsilon) genes and pseudogenes (psi) to the previously described fetal (gamma) and adult (beta) genes is as follows: 5'-epsilon 3-epsilon 4-psi 3-beta-epsilon 1-epsilon 2-psi 1-psi 2-gamma-3'. Present data indicate that, like that of the goat, the fetal and adult genes arose via block duplication of an ancestral four-gene set: epsilon-epsilon-psi-beta. This duplication event preceded the divergence of cows and goats, which occurred greater than or equal to 18-20 Myr ago. However, cows do not have the additional four-gene block containing a preadult/stress globin gene (beta C). Furthermore, the cow fetal cluster contains an extra beta-like pseudogene, which apparently arose by a small-scale duplication. The fixation of this duplication may indicate a possible evolutionary role for pseudogenes.

Ruminant globin gene structures suggest an evolutionary role for Alu-type repeats.

Bovine fetal and adult globin genes were cloned and subjected to DNA sequence analysis. Both of these genes contained insertions of Alu-type repetitive DNA within their introns. Comparison of cow and goat beta-type globin genes indicates that intragenic DNA insertions played a role in their evolution. These data support the theory that Alu-type repeats maintain genetic diversity by inhibiting gene conversion.

Fetal hemoglobin production in adult baboons by 5-azacytidine or by phenylhydrazine-induced hemolysis is associated with hypomethylation of globin gene DNA.

Nucleoside analog were used to stimulate fetal hemoglobin synthesis in baboons. Only those nucleoside analogs (5-azacytidine and 2'-deoxy-5-azacytidine) that blocked DNA methylation caused large increases in Hb F levels. DNA extracted from bone marrow samples of these animals was cleaved with MspI or HpaII restriction enzymes and analyzed by Southern blot hybridization. Results of these experiments strongly support the hypothesis that hypomethylation of CCGG sequences in the gamma-gene region is a condition of gamma-gene expression. This also applies to Hb F elevations resulting from erythropoietic stress due to phenylhydrazine-induced hemolytic anemia.

Total and spectral solar irradiance measured at ground surface.

This paper presents the results of a series of total and spectral solar irradiance measurements made at ground surface (Table Mountain Facility, Calif., altitude 2.18 km). The spectral irradiance data are presented for the 0.3-3.0-microm spectral region for air mass 1.5.

Structural analysis of interspersed repetitive polymerase III transcription units in human DNA.

The nucleotide sequences of two cloned fragments of human DNA which function as templates for RNA polymerase III in vitro confirm their identities as members of the Alu family of human interspersed repetitive DNA sequences (1,2). The interspersed and repetitive nature of these sequences in the genome was demonstrated by hybridization of nick-translated DNA from one of these clones to total genomic DNA and to DNA of individual random clones from a lambda Ch4A-based human genomic library. Short, direct terminal repeats of non-conserved sequence flank the 300 nucleotide Alu family conserved sequence. Within the Alu family sequence is found a 40-nucleotide region which is directly repeated 135 nucleotides downstream. This 40 nucleotide sequence is found once in the murine B1 interspersed repetitive sequence family (8). This and other evidence indicates that the human Alu family resembles a partial duplication of the murine B1 sequence.

Transcriptional analysis of interspersed repetitive polymerase III transcription units in human DNA.

The template for RNA polymerase III in vitro transcription found on the human DNA clone pJP53 was shown in the previous paper to enclose a member of the Alu famiy of interspersed repetitive DNA sequences. We have mapped this transcript onto its template in greater detail by comparison of the template DNA sequence to the base composition of the Tl ribonuclease digestion products of the in vitro transcript. We find that the 5' end of the transcript lies in close proximity to the 5' end of the conserved Alu family sequence as analyzed in the preceding paper. The 3' end of the transcript appears to terminate in a U-rich region beyond the region of Alu family sequence conservation. Analysis of cellular RNA by Northern blotting and hybridization with a DNA probe derived from another Alu family transcription template demonstrates abundant representation of sequences homologous to the reiterated DNA. Cytoplasmic, nonpolyadenylated RNA from human and murine cells contains a monodisperse, 300 nucleotide species, recently determined by Weiner (4) to be the 7S RNA. In contrast, the Alu-homologous transcripts are heterodisperse in mRNA and hnRNA, with the highest specific representation of Alu family sequences being found in oligo(dT)-retained hnRNA.