James W. Posakony

The structure and evolution of the human β-globin gene family

Argiris Efstratiadis Department of Biological Chemistry Harvard Medical School Boston, Massachusetts 02115 James W. Posakony, Tom Maniatis, Richard M. Lawn* and Catherine O’Connell+ Division of Biology California Institute of Technology Pasadena, California 91125 Richard A. Spritz, Jon K. DeRiel,# Bernard G. Forget and Sherman M. Weissman Departments of Genetics and Internal Medicine Yale University School of Medicine New Haven, Connecticut 06510 Jerry L. Slightom, Ann E. Blechl and Oliver Smithies Laboratory of Genetics University of Wisconsin Madison, Wisconsin 53706 Francisco E. Baralle, Carol C. Shoulders and Nicholas J. ProudfootQ MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 2QH, England Summary We present the results of a detailed comparison of the primary structure of human p-like globin genes and their flanking sequences. Among the se- quences located 5’ to these genes are two highly conserved regions which include the sequences ATA and CCAAT located 31 2 1 and 77 + 10 bp, respectively, 5’ to the mRNA capping site. Similar sequences are found in the corresponding locations in most other eucaryotic structural genes. Calcula- tion of the divergence times of individual @like globin gene pairs provides the first description of the evolutionary relationships within a gene family based entirely on direct nucleotide sequence com- parisons. In addition, the evolutionary relationship of the embryonic e-globin gene to the other human P-like globin genes is defined for the first time. Finally, we describe a model for the involvement of short direct repeat sequences in the generation of deletions in the noncoding and coding regions of B-like globin genes during evolution.

Mitochondrial DNA sequences in the nuclear genome of Strongylocentrotus purpuratus.

Two sea urchin embryo complementary DNA clones representing mitochondrial 16 S ribosomal RNA and cytochrome oxidase subunit I messenger RNA have been characterized. The cloned cDNAs are colinear with sea urchin mitochondrial DNA, and their identification is based on cross-hybridization with known restriction fragments of human mitochondrial DNA, and on nucleotide sequence determinations. The mitochondrial cDNA clones also displayed an unexpected reaction with specific genomic DNA sequences in gel blot hybridizations. Genomic phage lambda recombinants containing sequences hybridizing with the mitochondrial clones were isolated and the arrangement of these sequences was determined. The genomic region studied contains a sequence homologous with the 3 end of the mitochondrial 16 S rRNA gene, flanked on one side by what is possibly a complete copy of the cytochrome oxidase subunit I gene, and on the other by a duplication of a fragment of this gene. The nucleotide sequence divergence between the mitochondrial and nuclear homologues of the cytochrome oxidase subunit I gene varies for different regions of the gene, from about 13% to 25%, while there is about 8% sequence divergence between nuclear and mitochondrial versions of the 3 16S rRNA sequence. The structure of the genomic mitochondrial sequence homologues indicates that during sea urchin evolution there occurred a germ-line transposition of a fragment of the mitochondrial genome into the nuclear DNA, followed by rearrangements and single nucleotide substitutions.

Organization and expression of multiple actin genes in the sea urchin.

A set of at least 11 actin genes has been isolated from genomic recombinant deoxyribonucleic acid libraries of the sea urchin Strongylocentrotus purpuratus. Most of the isolates derive from a library which represents the genome of a single animal. There are at least five distinct types of sea urchin actin gene, some of which are represented by multiple copies in the genome. The actin gene types are distinguished by nonhomologous flanking sequences and intervening sequences, though the protein coding sequences appear in most cases to be quite similar. Eight of the 11 genes isolated have been recovered in lambda recombinants that contain two actin genes, linked at 5- to 9-kilobase distances. Restriction map overlaps suggest that the genome contains an array of at least three of these genes spaced over about 30 kilobases of deoxyribonucleic acid. In the linkage patterns observed, actin genes of diverse types were linked to each other. In early embryos, actin messenger ribonucleic acid (RNA) transcripts of 1.8 and 2.2 kilobases were found, and the longer of these transcripts was more prevalent in the maternal RNA of the egg. From RNA gel blot experiments, we conclude that the two transcripts derive from different actin gene types. Different repetitive sequences were located to either side of most of the actin genes, and in most observed cases the repeat sequences which were adjacent to actin genes of a given type were similar. The repeat sequences flanking the actin genes belonged to families which were transcribed, but those repeats in the neighborhood of the actin genes which have been investigated were not themselves represented in the stable RNAs of eggs or early embryos.

Interspersed sequence organization and developmental representation of cloned poly(A) RNAs from sea urchin eggs

A random primed complementary DNA (cDNA) clone library constructed from total maternal poly(A) RNA of sea urchin eggs was screened with two cloned genomic repetitive sequence probes. Sets of cDNA clones reacting with each of these repetitive sequences were recovered. Most of the cloned transcripts included both single copy and repeat sequence elements. Except for the shared repeat sequence element, both the repetitive and single copy regions of the members of each set of clones failed to crossreact. Single copy probes linked to the repeats on the cloned maternal RNAs are represented in an asymmetric manner. It follows that many different genomic members of a given dispersed repeat sequence family are represented in the maternal RNA. RNA gel blots carried out with several repeat probes display about 10 to 20 prominent maternal poly(A) RNAs containing transcripts of each repetitive sequence family. The interspersed maternal transcripts are 3000 to 15,000 bases in length. Maternal transcripts reacting with single copy probes derived from the cloned cDNAs persist during embryonic development, and in some cases appear to be augmented by similar, newly synthesized embryo transcripts. Two examples were found in which additional transcripts of different length appear at specific developmental stages. The transcribed single copy regions are highly polymorphic in the genomes of different individual sea urchins, and comparisons of closely related sea urchin species showed that both the prevalence and length of specific maternal transcripts change rapidly during evolution. Nucleotide sequences of two homologous repeat elements occurring on different cloned transcripts displayed translation stop codons in every possible reading frame. These repeat sequences display structural features suggesting that there has been evolutionary transposition into transcription units active during oogenesis. The repeat elements and their flanking single copy regions reside either in very long 3 or 5-terminal sequences, or in unprocessed intervening sequences in the maternal poly(A) RNA. These findings lead us to the proposal that the majority of the cytoplasmic poly(A) RNA in echinoderm eggs and early embryos is similar in form to RNAs that occur in the nucleus rather than to the messenger RNA of later cells.

Repetitive sequences of the sea urchin genome: Distribution of members of specific repetitive families☆

Three repetitive sequence families from the sea urchin genome were studied, each defined by homology with a specific cloned probe one to a few hundred nucleotides long. Recombinant λ-sea urchin DNA libraries were screened with these probes, and individual recombinants were selected that include genomic members of these families. Restriction mapping, gel blot, and kinetic analyses were carried out to determine the organization of each repeat family. Sequence elements belonging to the first of the three repeat families were found to be embedded in longer repeat sequences. These repeat sequences frequently occur in small clusters. Members of the second repeat family are also found in a long repetitive sequence environment, but these repeats usually occur singly in any given region of the DNA. The sequences of the third repeat are only 200 to 300 nucleotides long, and are generally terminated by single copy DNA, though a few examples were found associated with other repeats. These three repeat sequence families constitute sets of homologous sequence elements that relate distant regions of the DNA.

Repetitive sequences of the sea urchin genome: III. Nucleotide sequences of cloned repeat elements☆

The nucleotide sequences of eight randomly selected, cloned, repetitive sequence elements were determined. No homologies exist among the eight sequences that are sufficient to promote cross-reaction between them under standard conditions of measurement. Thus, each sequence is representative of a different repeat sequence family. Statistically significant but short (8 to 41 nucleotides) internal direct and inverse repetitions occupy a minor fraction of the sequence length in five of the eight repeat sequences. None contains internal reverse repeats sufficiently long to permit inclusion in the “foldback” DNA fraction. The general lack of internal sequence homology means that the sequence complexity of the eight clones is approximately equal to the length of the cloned inserts. Nucleotide sequences of three different members of one particular short interspersed repeat sequence family are also reported. Comparison of these sequences reveals that both the number and order of internal sequence subelements differ among family members. The results show that both fine-scale rearrangement and sequence divergence have occurred during the evolution of this repeat family.

Repetitive sequences of the sea urchin genome: II. Subfamily structure and evolutionary conservation

Members of three repetitive sequence families were isolated from recombinant λ-genome libraries, and were used to investigate sequence relationships within these families. Studies presented elsewhere show that members of all three repeat sequence families are transcribed tissue-specifically. The thermal stability of intrafamilial heteroduplexes was measured, and the extent of colinearity between related sequences was determined by restriction mapping, heteroduplex visualization, gel blot hybridization, and direct sequencing. One large and very divergent family, named 2108, was shown to consist of an assemblage of many small repeat sequence subfamilies. Each subfamily includes <40 members which are not contiguous in the genome but are very closely related colinear sequence elements several thousand nucleotides in length. The different 2108 subfamilies share only small sequence subelements, which in each subfamily occur in a different linear order and are surrounded by different sequences. A second divergent family consisting of short repetitive sequences, the 2109 family, includes many small internally homologous subfamilies as well. A third family, 2034, displays little internal sequence divergence and no apparent subfamily structure. The repeat sequence subfamilies may be biologically significant units of repetition. Thus specific 2108 subfamilies were shown to be evolutionary conserved to a remarkable degree. Highly homologous 2108 sequences were found shared among sea urchin species which diverged almost 200 million years ago, although only about 10% of the single copy DNA sequences of these species are now homologous enough to crossreact.

Molecular structure of maternal RNA

The presence of a stable maternal mRNA population in mature oocytes of many species is well established. In this paper we show that the mature egg contains, in addition to these mature mRNAs, a structurally more complex population of RNA transcripts. This latter class of RNA consists of polyadenylated transcripts of repetitive and nonrepetitive DNA elements covalently linked into long interspersed molecules. As much as seventy percent of the polyadenylated egg RNA of Xenopus laevis and Strongylocentrotus purpuratus is represented in this interspersed population. Most of the nonrepetitive DNA sequences represented in the mature mRNA population are also present in the interspersed RNA. These transcripts have an organization similar to that of somatic cell nuclear RNA. Data are presented that suggests some of these interspersed maternal transcripts are unprocessed precursor-like molecules. Some possible functions of this novel class of RNA during early development are discussed.

Transcripts of three mitochondrial genes in the RNA of sea urchin eggs and embryos

cDNA clones representing mitochondrial 16 S rRNA, and mRNAs for cytochrome oxidase I and an unidentified reading frame were used to measure the prevalence and stability of these transcripts in gastrula stage embryos. The 16 S rRNA is the most prevalent embryo poly(A) RNA, and is synthesized about four times more rapidly than is the mRNA for cytochrome oxidase. The relative prevalence of the two mRNAs is largely determined by their turnover rates.