Biomedical subjects

T H Eickbush

Publications and source records attributed to T H Eickbush.

At least 19 recordsLinked to original sources

PubMed1992

Molecular characterization of repetitive DNA sequences from a B chromosome.

In the parasitic wasp Nasonia vitripennis, certain males carry a B chromosome, called PSR (paternal sex ratio), which causes the compaction and subsequent loss of the paternal chromosomes in fertilized eggs. Because Nasonia are haplo-diploid, this leads to the production of all-male broods. Three families (PSR2, PSR18, PSR22) of related, tandemly repetitive DNAs were shown to be present solely on the PSR chromosome. These three families shared two conserved, palindromic DNA sequences, which may play a role in either PSR function or amplification of the tandem arrays. The tandem repeat family NV79 was determined to be present on the PSR chromosome as well as on at least one of the A chromosomes. This shared repeat as well as two repeat families (NV85, NV126) that were localized on the A chromosomes were detected in two sibling species of N. vitripennis. NV79 and NV126 were also found in the more distantly related species, Trichomalopsis dubius.

Animals↗

PubMed1992

Turnover of R1 (type I) and R2 (type II) retrotransposable elements in the ribosomal DNA of Drosophila melanogaster.

R1 and R2 are distantly related non-long terminal repeat retrotransposable elements each of which inserts into a specific site in the 28S rRNA genes of most insects. We have analyzed aspects of R1 and R2 abundance and sequence variation in 27 geographical isolates of Drosophila melanogaster. The fraction of 28S rRNA genes containing these elements varied greatly between strains, 17-67% for R1 elements and 2-28% for R2 elements. The total percentage of the rDNA repeats inserted ranged from 32 to 77%. The fraction of the rDNA repeats that contained both of these elements suggested that R1 and R2 exhibit neither an inhibition of nor preference for insertion into a 28S gene already containing the other type of element. Based on the conservation of restriction sites in the elements of all strains, and sequence analysis of individual elements from three strains, nucleotide divergence is very low for R1 and R2 elements within or between strains (less than 0.6%). This sequence uniformity is the expected result of the forces of concerted evolution (unequal crossovers and gene conversion) which act on the rRNA genes themselves. Evidence for the role of retrotransposition in the turnover of R1 and R2 was obtained by using naturally occurring 5' length polymorphisms of the elements as markers for independent transposition events. The pattern of these different length 5' truncations of R1 and R2 was found to be diverse and unique to most strains analyzed. Because recombination can only, with time, amplify or eliminate those length variants already present, the diversity found in each strain suggests that retrotransposition has played a critical role in maintaining these elements in the rDNA repeats of D. melanogaster.

Animals↗

PubMed1992

Transposing without ends: the non-LTR retrotransposable elements.

Transposable elements have been discovered in animals, plants, fungi, and protozoans which contain open reading frames similar to the gag and pol genes of retroviruses and retrotransposons but which lack long terminal repeats (LTRs). Recent experiments have shown that these non-LTR elements [also called poly(A) type and LINE-like elements] encode functional reverse transcriptase and replicate via an RNA intermediate. Based on phylogenetic analysis of their encoded reverse transcriptase sequences, the non-LTR retrotransposons are the likely progenitors of retroviruses and LTR retrotransposons. Because retroviruses and LTR retrotransposons depend upon their LTRs for key steps in both transcription and integration, the mechanisms utilized by the non-LTR retrotransposons must be fundamentally different. Internal promoter sequences have been found in several non-LTR elements that initiate transcription upstream at the first nucleotide. Current models for retrotransposition of non-LTR elements propose that the 3' ends of staggered nicks at the chromosomal insertion site serve as primers for first- and second-staggered nicks at the chromosomal insertion site serve as primers for first- and second-strand synthesis from the RNA template. These models suggest that the enzymatic machinery of non-LTR elements is likely to be responsible for the integration of SINEs and processed pseudogenes.

Animals↗

PubMed1991

Retrotransposable elements R1 and R2 interrupt the rRNA genes of most insects.

A large number of insect species have been screened for the presence of the retrotransposable elements R1 and R2. These elements integrate independently at specific sites in the 28S rRNA genes. Genomic blots indicated that 43 of 47 insect species from nine orders contained insertions, ranging in frequency from a few percent to greater than 50% of the 28S genes. Sequence analysis of these insertions from 8 species revealed 22 elements, 21 of which corresponded to R1 or R2 elements. Surprisingly, many species appeared to contain highly divergent copies of R1 and R2 elements. For example, a parasitic wasp contained at least four families of R1 elements; the Japanese beetle contained at least five families of R2 elements. The presence of these retrotransposable elements throughout Insecta and the observation that single species can harbor divergent families within its rRNA-encoding DNA loci present interesting questions concerning the age of these elements and the possibility of cross-species transfer.

Amino Acid Sequence↗

PubMed1991

Sequence identity in an early chorion multigene family is the result of localized gene conversion.

The multigene families that encode the chorion (eggshell) of the silk moth, Bombyx mori, are closely linked on one chromosome. We report here the isolation and characterization of two segments, totaling 102 kb of genomic DNA, containing the genes expressed during the early period of choriogenesis. Most of these early genes can be divided into two multigene families, ErA and ErB, organized into five divergently transcribed ErA/ErB gene pairs. Nucleotide sequence identity in the major coding regions of the ErA genes was 96%, while nucleotide sequence identity for the ErB major coding regions was only 63%. Selection pressure on the encoded proteins cannot explain this difference in the level of sequence conservation between the ErA and ErB gene families, since when only fourfold redundant codon positions are considered, the divergence within the ErA genes is 8%, while the divergence within the ErB genes (corrected for multiple substitutions at the same site) is 110%. The high sequence identity of the ErA major exons can be explained by sequence exchange events similar to gene conversion localized to the major exon of the ErA genes. These gene conversions are correlated with the presence of clustered copies of the nucleotide sequence GGXGGX, encoding paired glycine residues. This sequence has previously been correlated with gradients of gene conversion that extend throughout the coding and noncoding regions of the High-cysteine (Hc) chorion genes of B. mori. We suggest that the difference in the extent of the conversion tracts in these gene families reflects a tendency for these recombination events to become localized over time to the protein encoding regions of the major exons.

Animals↗

PubMed1990

Type I (R1) and type II (R2) ribosomal DNA insertions of Drosophila melanogaster are retrotransposable elements closely related to those of Bombyx mori.

Approximately 50% of the ribosomal DNA (rDNA) units of Drosophila melanogaster are inactivated by two different 28 S RNA ribosomal gene insertions (type I and type II). We present here the nucleotide sequence of complete type I and type II elements. Conceptual translation of these sequences revealed open reading frames (ORFs) encoding amino acid residues conserved in all retrotransposable elements. Full-length type I elements are 5.35 x 10(3) base-pairs in length and contain two overlapping ORFs. The smaller ORF (471 amino acid residues) has similarity to gag genes, while the larger ORF (1021 residues) has similarity to pol genes. Full-length type II elements are 3.6 x 10(3) base-pairs and contain one large ORF (1056 residues) that appears to represent a gag-pol fusion. Type I and type II elements are similar in structure, in the proteins they encode, and in insertion specificity to the R1Bm and R2Bm retrotransposable elements of Bombyx mori. We suggest that the D. melanogaster elements be called R1Dm and R2Dm, to reflect their structure as retrotransposons. Comparison of the R1 and R2 elements from these two widely different species revealed regions of the ORF that are likely to play an important role in the propagation of the elements. Four distinct regions of sequence conservation separated by regions of little or no sequence similarity were detected for both the R1 and R2 elements: (1) cysteine motifs of the gag gene, with three such motifs for R1 and one motif for R2; (2) a reverse transcriptase domain; (3) an integrase domain located carboxyl terminal to the reverse transcriptase region; and (4) a small region amino terminal to the reverse transcriptase domain, whose function is not known. The level of identity of the amino acid residues for these segments is 28 to 34% between the R1 elements, and 34 to 39% for the R2 elements. Finally, it may be predicted that the mechanism of unequal crossover might eventually eliminate R1 and R2 from the rDNA locus. The long history of selection at the protein level exhibited by these elements indicates that it is their active transposition that maintains them in the locus. The high level of sequence homogeneity between copies of each element within the same species is consistent with the high turnover rate expected to result from these processes.

Amino Acid Sequence↗

PubMed1990

Origin and evolution of retroelements based upon their reverse transcriptase sequences.

To study the evolutionary relationship of reverse transcriptase (RT) containing genetic elements, a phylogenetic tree of 82 retroelements from animals, plants, protozoans and bacteria was constructed. The tree was based on seven amino acid domains totalling 178 residues identified in all RTs. We have also identified these seven domains in the RNA-directed RNA polymerases from various plus-strand RNA viruses. The sequence similarity of these RNA polymerases to RT suggests that these two enzymes evolved from a common ancestor, and thus RNA polymerase can be used as an outgroup to root the RT tree. A comparison of the genetic organization of the various RT containing elements and their position on the tree allows several inferences concerning the origin and evolution of these elements. The most probable ancestor of current retroelements was a retrotransposable element with both gag-like and pol-like genes. On one major branch of the tree, organelle and bacterial sequences (e.g. group II introns and bacterial msDNA) appear to have captured the RT sequences from retrotransposons which lack long terminal repeats (LTRs). On the other major branch, acquisition of LTRs gave rise to two distinct groups of LTR retrotransposons and three groups of viruses: retroviruses, hepadnaviruses and caulimoviruses.

Amino Acid Sequence↗

PubMed1989

Gene evolution and regulation in the chorion complex of Bombyx mori. Hybridization and sequence analysis of multiple developmentally middle A/B chorion gene pairs.

Twenty-two pairs of chorion genes belonging to the A and B multigene families have been characterized and mapped within two segments of a 320 kb (1 kb = 10(3) bases or base-pairs) chromosomal walk in the domesticated silkmoth Bombyx mori. Eighteen of the gene pairs belong to two groups that are typified by the previously characterized A/B.L12 and A/B.L11 chorion gene pairs, and are defined by two respective types of short (approx. 280 base-pairs) bidirectional promoter sequences. In the chromosome, the L12-like and L11-like pairs are interspersed with each other and with the remaining four gene pairs, which have unrelated promoter sequences. We have sequenced the promoter regions and adjacent small exons of all L12-like and L11-like A and B genes in the walk. The L12-like promoters are highly conserved, whereas L11-like promoters are somewhat more variable. Reconsideration of previous data on RNA accumulation and disappearance during choriogenesis, in the light of the sequences, indicates that L12-like genes are developmentally early-middle, while L11-like genes correspond to two developmental subgroups, middle I and middle II. Sequence comparisons of all these promoters, as well as the previously characterized promoters of the developmentally late HcA and HcB genes, identify short elements of possible regulatory significance. The sequences, as well as extensive cross-hybridization analysis with short probes derived from the reference A/B.L12 gene pair, under carefully controlled conditions of stringency, indicate the occurrence of sequence transfers among A or B genes. These sequence transfers, which could result from gene conversions or unequal crossovers, are less abundant than in the HcA and HcB families, but do result in a patchwork of similarities and differences in the A and B genes. The transfers appear to be least frequent between the moderately divergent A genes that belong to different temporal classes, while the L12-like and L11-like B genes appear to be extensively homogenized in sequence.

Animals↗

PubMed1988

H2a-specific proteolysis as a unique probe in the analysis of the histone octamer.

We have utilized the H2a-specific protease as a unique probe to investigate the nature of the interactions between the protein subunits which form the core histone octamer. Upon incubation in high ionic strength media this protease, normally found tightly associated with isolated calf thymus chromatin, releases the 15 COOH-terminal amino acids of histone H2a by specifically cleaving the H2a polypeptide between Val114 and Leu115, yielding cleaved H2a (cH2a) and a free pentadecapeptide (Eickbush, T. H., Watson, D. K., and Moudrianakis, E. N. (1976) Cell 9, 785-792). We find that removal of this pentadecapeptide results in a marked dissociation of the octamer into its H2a:H2b dimer and H3:H4 tetramer subunits. Reconstitution experiments indicate that cH2a is capable of forming a dimer with H2b, but this cH2a:H2b dimer has a substantially lower affinity for the H3:H4 tetramer than native H2a:H2b dimer. Kinetic studies of H2a cleavage in high ionic strength solutions demonstrate that H2a molecules in the octamer are relatively resistant to proteolytic attack compared to H2a molecules in the dimer. The extent of this resistance, in response to various experimental parameters, is directly correlated to the strength of interaction between the H2a:H2b dimer and H3:H4 tetramer subunits. These reconstitution and kinetic experiments suggest that the histone domains proximal to the H2a cleavage site have an important function in maintaining the association of the histone octamer subunits.

Animals↗

PubMed1988

Ribosomal DNA insertion elements R1Bm and R2Bm can transpose in a sequence specific manner to locations outside the 28S genes.

A fraction of the ribosomal 28S genes in some insects are interrupted at specific sites by insertion elements R1 and R2 (also called Type I and II). These elements contain long open-reading frames with homology to reverse transcriptase. We have identified in the silkmoth, Bombyx mori, copies of these elements which have inserted into sites outside the ribosomal DNA (rDNA) units. The 3' ends of all "non-rDNA" elements are identical to the elements within the 28S genes; however their 5' ends are often truncated. Each non-rDNA copy has inserted into sequences that exhibit similarity to their target sites in the 28S gene. We also demonstrate by genomic blot analysis of different strains of B. mori that insertions of R1 and R2 outside the rDNA units have been infrequent, while considerable turnover of elements has occurred within the rDNA locus. One race of B. mori has lost all copies of R1 from its rDNA units, while retaining normal levels of R2. The level of both R1 and R2 have significantly increased in a tissue culture line. These findings add considerable support to the model that R1 and R2 are retrotransposable elements that utilize sequence specific endonucleases in their integration into the genome.

Animals↗

PubMed1988

Functional expression of a sequence-specific endonuclease encoded by the retrotransposon R2Bm.

A fraction of the 28S ribosomal genes in certain insect species is interrupted by the insertion elements R1 and R2. These two elements from the silkworm Bombyx mori (R1Bm and R2Bm) are retrotransposons capable of transposing in a highly sequence-specific manner. We report here the functional expression in E. coli of the entire single open reading frame of R2Bm and show that it encodes a double-stranded endo-nuclease (integrase) that can specifically cleave the 28S gene at the R2 insertion site. The resulting cleavage is a 4 bp staggered 5' overhang. Deletion analysis of the 28S gene revealed that the DNA sequence required for specific cleavage is asymmetric with respect to the actual insertion (cleavage) site, with fewer than 10 bp required at one side and at least 24 bp at the other side of the site. A model is proposed based on these and previous data to account for the sequence-specific integration of the R2 retrotransposon.

Animals↗

PubMed1988

A "selfish" B chromosome that enhances its transmission by eliminating the paternal genome.

In the parasitic wasp, Nasonia vitripennis, males are haploid and usually develop from unfertilized eggs, whereas females are diploid and develop from fertilized eggs. Some individuals in this species carry a genetic element, termed psr (paternal sex ratio), which is transmitted through sperm and causes condensation and subsequent loss of paternal chromosomes in fertilized eggs, thus converting diploid females into haploid males. In this report the psr trait was shown to be caused by a supernumerary chromosome. This B chromosome contains at least three repetitive DNA sequences that do not cross-hybridize to each other or to the host genome. The psr chromosome apparently produces a trans-acting product responsible for condensation of the paternal chromosomes, but is itself insensitive to the effect. Because the psr chromosome enhances its transmission by eliminating the rest of the genome, it can be considered the most "selfish" genetic element yet described.

Animals↗

PubMed1988

Organization and expression of three genes from the silkmoth early chorion locus.

Chorion genes in the silkmoth Bombyx mori are clustered in a small region of one chromosome. Genes are grouped within this locus according to their expression in either early, middle, or late stages of choriogenesis. The entire set of late genes and a large fraction of the middle genes have been cloned and extensively characterized. We report here the first molecular characterization of a set of early chorion genes clustered within a 22.5-kb region. Transcripts homologous to these genes appear in the very first choriogenic follicle but disappear with different kinetics. One of the three early genes, 5H4, is the first example of a chorion gene which is not a member of a multigene family, or divergently paired with another chorion gene. The other two early genes, (ErA.1 and ErB.1) are divergently paired with each other in the manner of the middle and late chorion genes. The common 5' region between ErA.1 and ErB.1 is significantly shorter and does not contain certain sequence elements shared by the highly conserved 5' regions of middle and late chorion gene pairs. This evidence taken together with the fact that 5H4 is unpaired indicates that the early genes may utilize a regulatory mechanism somewhat different from the middle and late gene families.

Amino Acid Sequence↗

PubMed1988

Gene conversions can generate sequence variants in the late chorion multigene families of Bombyx mori.

The 140-kbp late chorion locus of Bombyx mori strain 703 contains 15 divergently oriented gene pairs encoding the high cysteine (Hc) eggshell proteins. Sequence homology is approximately 91% for the 2-kb region of each gene pair, including the 5' flanking region, intron and exons. The homology rapidly disappears within a few hundred basepairs of the 3' end of most genes. Here we present the results of the nucleotide sequence and genomic blot comparison of Hc genes from different races of B. mori. Comparison of the nucleotide sequences of the same gene pair in two different races reveals that most of the nucleotide differences occur in clusters or patches and correspond to sequences present in other Hc genes in the locus. The number of nucleotide differences that have accumulated in the highly conserved regions of the gene pair (2.3/100 bp), most of which are attributable to patchwork exchanges, is significantly higher than the number of differences in the poorly conserved 3' flanking regions (0.6/100 bp), due primarily to new mutations. These data are consistent with a gene conversion process, which in the short-term generates new combinations of sequence variants, but in the long-term results in concerted evolution. Genomic blot analyses of different geographical races of B. mori reveal that there is variation in the number of Hc gene pairs (14-19 gene pairs), indicating that unequal crossovers also occur in the locus.

Animals↗

PubMed1988

Similarity of reverse transcriptase-like sequences of viruses, transposable elements, and mitochondrial introns.

Sequences similar to reverse transcriptase (RT) of retroviruses have been found in certain DNA viruses, mitochondrial intron sequences, and a wide variety of transposable elements. While total amino acid similarity between these diverse elements is quite low, we have identified seven regions, consisting of 182 amino acids, that are common to all elements. Highly conserved residues identified in each of these regions are diagnostic for the identification and alignment of these and for future RT-like sequences. Using both the neighbor-joining and the unweighted-pair-group methods, we have derived a probable phylogenetic tree for all RT-containing elements. These elements can be divided into two major groups. Retroviruses and DNA viruses whose propagation involves an RNA intermediate are grouped with a series of transposable elements containing long terminal repeats (LTRs). The second group is made up of RT-containing sequences of fungal mitochondrial introns and a series of transposable elements that lack LTRs. The transposable elements, copia and Ty, were found to be the most difficult to position on the phylogenetic tree, as a result of their higher rate of sequence divergence. The data are most consistent with their being distant members of the LTR group (retroviruses/LTR retrotransposons).

Amino Acid Sequence↗

PubMed1988

The site-specific ribosomal DNA insertion element R1Bm belongs to a class of non-long-terminal-repeat retrotransposons.

Two types of insertion elements, R1 and R2 (previously called type I and type II), are known to interrupt the 28S ribosomal genes of several insect species. In the silkmoth, Bombyx mori, each element occupies approximately 10% of the estimated 240 ribosomal DNA units, while at most only a few copies are located outside the ribosomal DNA units. We present here the complete nucleotide sequence of an R1 insertion from B. mori (R1Bm). This 5.1-kilobase element contains two overlapping open reading frames (ORFs) which together occupy 88% of its length. ORF1 is 461 amino acids in length and exhibits characteristics of retroviral gag genes. ORF2 is 1,051 amino acids in length and contains homology to reverse transcriptase-like enzymes. The analysis of 3' and 5' ends of independent isolates from the ribosomal locus supports the suggestion that R1 is still functioning as a transposable element. The precise location of the element within the genome implies that its transposition must occur with remarkable insertion sequence specificity. Comparison of the deduced amino acid sequences from six retrotransposons, R1 and R2 of B. mori, I factor and F element of Drosophila melanogaster, L1 of Mus domesticus, and Ingi of Trypanosoma brucei, reveals a relatively high level of sequence homology in the reverse transcriptase region. Like R1, these elements lack long terminal repeats. We have therefore named this class of related elements the non-long-terminal-repeat (non-LTR) retrotransposons.

Amino Acid Sequence↗

PubMed1987

The site-specific ribosomal insertion element type II of Bombyx mori (R2Bm) contains the coding sequence for a reverse transcriptase-like enzyme.

Two classes of DNA elements interrupt a fraction of the rRNA repeats of Bombyx mori. We have analyzed by genomic blotting and sequence analysis one class of these elements which we have named R2. These elements occupy approximately 9% of the rDNA units of B. mori and appear to be homologous to the type II rDNA insertions detected in Drosophila melanogaster. Approximately 25 copies of R2 exist within the B. mori genome, of which at least 20 are located at a precise location within otherwise typical rDNA units. Nucleotide sequence analysis has revealed that the 4.2-kilobase-pair R2 element has a single large open reading frame, occupying over 82% of the total length of the element. The central region of this 1,151-amino-acid open reading frame shows homology to the reverse transcriptase enzymes found in retroviruses and certain transposable elements. Amino acid homology of this region is highest to the mobile line 1 elements of mammals, followed by the mitochondrial type II introns of fungi, and the pol gene of retroviruses. Less homology exists with transposable elements of D. melanogaster and Saccharomyces cerevisiae. Two additional regions of sequence homology between L1 and R2 elements were also found outside the reverse transcriptase region. We suggest that the R2 elements are retrotransposons that are site specific in their insertion into the genome. Such mobility would enable these elements to occupy a small fraction of the rDNA units of B. mori despite their continual elimination from the rDNA locus by sequence turnover.

Amino Acid Sequence↗

PubMed1986

A highly reiterated family of transcribed oligo(A)-terminated, interspersed DNA elements in the genome of Bombyx mori.

A library of low Cot DNA (Cot is the molar concentration of DNA times the incubation time in seconds) from Bombyx mori was used to isolate five independent clones of highly reiterated sequences from the genome of this organism. Sequence analysis revealed that all five clones belong to a single family of repetitive DNA elements, which we have named Bm1, and whose reiteration frequency is approximately 2.3 X 10(4) copies per haploid genome. Probing of a Bombyx genomic library (in lambda phage) with a Bm1 clone reveals that this repetitive sequence is dispersed throughout the genome. The pattern of interspersion was confirmed by Southern blot mapping of a large (270 X 10(3) base-pairs) domain of the chorion locus of Bombyx, where at least 13 independent regions were found to hybridize to Bm1. Four additional Bm1 elements have been sequenced from a 4.8 X 10(3) base-pair genomic fragment containing an early chorion gene. Two of these four elements are bounded by short (4 to 12 base-pairs) direct repeats. The nine Bm1 elements which have been sequenced are greater than 88% homologous to each other, and tend to fall in at least two size classes (253 base-pairs and 450 base-pairs). Seven of the nine Bm1 elements have a short 6 to 10 base-pair oligo(A) sequence at the 3' end. A sequence of about 29 base-pairs at the 3' end, including the oligo(A), shows 86% homology to the equivalent 3'-terminal domain of human Alu family repetitive elements. A 129 base-pair domain at the 5' end of Bm1 shows 66% homology to a Drosophila valine transfer RNA gene; thus the 5' end of Bm1 may contain the split internal RNA polymerase III promoter that is characteristic of most transcribed tRNA-like retroposons. Dot-blot analysis of Bombyx RNA shows that Bm1 DNA is indeed transcribed, and that the transcripts are well-represented in the total RNA of an ovarian-derived permanent cell line and posterior silk glands early in the fifth instar, but are less abundant in the RNA of pupae or silk glands late in the fifth instar.

Animals↗