Terminally redundant sequences in cellular intracisternal A-particle genes.

The sequences coding for intracisternal A-particle RNA form a family of related but not identical genetic elements which are present in 650 to 1,000 copies within the mouse genome. We showed that different intracisternal A-particle genes had a terminally redundant sequence of about 400 base pairs, one-half of which arose from the 3' end of the intracisternal A-particle RNA. A second portion of the redundant region did not contain 3-related sequences and was probably derived from the 5' end of intracisternal A-particle RNA. Thus, there were endogenous intracisternal A-particle genes in the cellular DNA-3'-5'--3'-5'-cellular DNA configuration identified for type B and C retroviruses. This indicated that the initial integration of intracisternal A-particle genes into the Mus musculus genome occurred by the same mechanism as the integration of other retroviruses. Two types of heterogeneity were identified among the 5' sequences of the two genes.     

Predicting mammalian SINE subfamily activity from A-tail length

Based on previous observations that newly inserted LINEs and SINEs have particularly long 3' A-tails, which shorten rapidly during evolutionary time, we have analyzed the rat and mouse genomes for evidence of recently inserted SINEs and LINEs. We find that the youngest predicted subfamilies of rodent identifier (ID) elements, a rodent-specific SINE derived from tRNA(Ala), are preferentially associated with A-tails over 50 bases in the rat genome, as predicted. Furthermore, these studies detected a subfamily of ID elements that has made over 15,000 copies that is younger than any previously reported ID subfamily. We use PCR analysis of genomic loci to demonstrate that all subfamily members tested inserted after the divergence of Rattus norvegicus from Rattus rattus. We also found evidence that the rodent B1 family of elements is much more active currently in mouse than in rat. These data provide useful estimates of recent activity from all of the mammalian retrotransposons, as well as allowing identification of the most recent insertions for use as population and speciation markers in those species. Both the current rat ID and mouse B1 elements that are active have small, specific interruptions in their 3' A-tail sequences. We suggest that these interruptions stabilize the length of the A-tails and contribute to the activity of these subfamilies. We present a model in which the dynamics of the 3' A-tail may be a central controlling factor in SINE activity.     

Structural analysis of type II variants within the mouse intracisternal A-particle sequence family.

Intracisternal A-particle (IAP) elements are present in multiple copies in the mouse and other rodent genomes. The bulk of this sequence family in Mus musculus consists of 7 Kb long elements, but the majority of IAP sequences involved in known transpositions have been deleted forms. The present study describes a subset of deleted IAP sequences (type II IAP) characterized by insertion of a particular short sequence element (AIIins). AIIins are interspersed and the majority occur as part of the type II IAP elements in the mouse genome. AIIins sequences are absent or in low copy number outside Mus musculus. We have isolated clones containing AIIins from a mouse genomic DNA library and have sequenced three isolates of AIIins and their surrounding IAP sequences to define the detailed structure of type II elements. AIIins are 272, 268 and 264 bp long and 90% homologous in sequence. They are bracketed by 9 bp duplications, suggesting they may be inserted elements. A 75 bp region containing a core enhancer sequence is repeated at the 5' end in type II IAP elements. Insertion into the IAP genome, with potential to encode an integrase function, may have played a role in the amplification of AIIins.     

A highly conserved repeated DNA element located in the chromosome of Streptococcus pneumoniae.

We report the discovery of a group of highly conserved DNA sequences located, in those cases studied, within intergenic regions of the chromosome of the Gram positive Streptococcus pneumoniae. The S. pneumoniae genome contains about 25 of these elements called BOX. From 5' to 3', BOX elements are composed of three subunits (boxA, boxB, and boxC) which are 59, 45 and 50 nucleotides long, respectively. BOX elements containing one, two and four copies of boxB have been observed; boxB alone was also detected in one instance. These elements are unrelated to the two most thoroughly documented families of repetitive DNA sequences present in the genomes of enterobacteria. BOX sequences have the potential to form stable stem-loop structures and one of these, at least, is transcribed. Most of these elements are located in the immediate vicinity of genes whose product has been implicated at some stage in the process of genetic transformation or in virulence of S. pneumoniae. This location raises the intriguing possibility that BOX sequences are regulatory elements shared by several coordinately controlled genes, including competence-specific and virulence-related genes.     

Genomic structure and evolutionary conservation of the tyrosinase gene family from Fugu

The tyrosinase gene family encompasses three members, tyrosinase, tyrosinase-related protein 1 (Tyrp1) and dopachrome tautomerase (Dct), which encode for proteins implicated in melanin synthesis. In human and mouse, genomic organization is known for all three genes, revealing common features of regulatory elements and of exon/intron structure. We have set out to identify the complete family from a more primitive vertebrate, the pufferfish Fugu (Takifugu rubripes), which is characterized by a compact genome. We had recently isolated and characterized the Fugu tyrosinase gene (Genesis 28 (2000) 99-105). We now report the isolation and characterization of the two other members of the family, Tyrp1 and Dct. Regulatory sequences from these genes function in mouse pigment cells and are able to mediate reporter gene expression. Our results demonstrate the existence of all three tyrosinase family members in teleosts and underline the evolutionary conservation of the pigmentary system.     

Identification of a conserved sequence in the non-coding regions of many human genes.

We have analyzed a sequence of approximately 70 base pairs (bp) that shows a high degree of similarity to sequences present in the non-coding regions of a number of human and other mammalian genes. The sequence was discovered in a fragment of human genomic DNA adjacent to an integrated hepatitis B virus genome in cells derived from human hepatocellular carcinoma tissue. When one of the viral flanking sequences was compared to nucleotide sequences in GenBank, more than thirty human genes were identified that contained a similar sequence in their non-coding regions. The sequence element was usually found once or twice in a gene, either in an intron or in the 5' or 3' flanking regions. It did not share any similarities with known short interspersed nucleotide elements (SINEs) or presently known gene regulatory elements. This element was highly conserved at the same position within the corresponding human and mouse genes for myoglobin and N-myc, indicating evolutionary conservation and possible functional importance. Preliminary DNase I footprinting data suggested that the element or its adjacent sequences may bind nuclear factors to generate specific DNase I hypersensitive sites. The size, structure, and evolutionary conservation of this sequence indicates that it is distinct from other types of short interspersed repetitive elements. It is possible that the element may have a cis-acting functional role in the genome.     

A new family of interspersed repetitive DNA sequences in the mouse genome

Repetitive DNA sequences near immunoglobulin genes in the mouse genome (Steinmetz et al., 1980a,b) were characterized by restriction mapping and hybridization. Six sequences were determined that turned out to belong to a new family of dispersed repetitive DNA. From the sequences, which are called R1 to R6, a 475 base-pair consensus sequence was derived. The R family is clearly distinct from the mouse B1 family (Krayev et al., 1980). According to saturation hybridization experiments, there are about 100,000 R sequences per haploid genome, and they are probably distributed throughout the genome. The individual R sequences have an average divergence from the consensus sequence of 12.5%, which is largely due to point mutations and, among those, to transitions. Some R sequences are severly truncated. The R sequences extend into A-rich sequences and are flanked by short direct repeats. Also, two large insertions in the R2 sequence are flanked by direct repeats. In the neighbourhood of and within R sequences, stretches of DNA have been identified that are homologous to parts of small nuclear RNA sequences. Mouse satellite DNA-like sequences and members of the B1 family were also found in close proximity to the R sequences. The dispersion of R sequences within the mouse genome may be a consequence of transposition events. The possible role of the R sequences in recombination and/or gene conversion processes is discussed.     

Molecular cloning of retrovirus-like genes present in multiple copies in the Syrian hamster genome.

Endogenous retrovirus-like sequences homologous to intracisternal type-A particle (IAP) genes, which are present in the inbred mouse (Mus musculus) genome, were cloned from a Syrian hamster gene library. A typical hamster IAP gene was 7 kb long and segments homologous to long terminal repeat (IAP) sequences present in Mus musculus IAP genes were located at both ends of the gene. Contrary to the pattern found in the Mus musculus IAP genes, the organization of the cloned hamster IAP genes was not markedly polymorphic and deletion was not observed among these cloned genes. A sequence about 0.8 kb long and located close to the 3' end of the hamster IAP gene was well conserved in both IAP gene families, although they showed less overall homology with one another. The reiteration frequency of the hamster IAP genes was calculated to be 950 copies per haploid genome. Since such IAP genes with the above properties were not found in the genome of the Chinese hamster, whose progenitors diverged from those of the Syrian hamster about 7.5 Myr ago, the integration of a huge number of Syrian hamster IAP genes must have occurred subsequent to such divergence.     

Comparison of the sequence organization of related retrovirus-like multigene families in three evolutionarily distant rodent genomes.

Sequences related to mouse intracisternal A-particle (IAP) genes have been isolated from rat and Syrian hamster gene libraries as recombinants in lambda phage. The sequences are moderately reiterated in both these genomes but their sequence organization in the hamster genome is different from that in the rat genome. Restriction analysis and electron microscopy indicate that the Syrian hamster IAP sequences represent a family of relatively homogeneous well-conserved units; in this, they resemble the mouse IAP genes. The rat sequences, in contrast, are heterogeneous. Both the hamster and rat IAP sequences contain regions homologous to mouse IAP genes interspersed with regions of apparent non-homology. The interspersed regions range in size from 0.5-1.0 kilobases (Kb). The regions of homology among the mouse, rat and Syrian hamster IAP sequences have been mapped to a 5-6 Kb internal region on the mouse IAP genes. Mouse IAP long terminal repeat (LTR) sequences were not detected in the rat and Syrian hamster genomes. We used the thermal stability of hybrids between cloned and genomic IAP sequences to measure family homogeneity. Mouse and Syrian hamster IAP sequences are homogeneous by this criterion, but the rat IAP sequences are heterogeneous with a Tm 6 degrees C below the self-hybrid. The contrasting organization of IAP-related elements in the genomes of these rodents indicates that amplification or homogenization of this sequence family has occurred independently and at different periods of time during their evolution.     

Determination of a functional ancestral sequence and definition of the 5' end of A-type mouse L1 elements

The complete nucleotide sequence of L1Md-A13, a 6372 base-pair (bp) member of the L1Md repetitive family isolated from a BALB/c mouse genomic DNA library, is reported. The nucleotide sequence of 4331 bp from the 5' end of L1Md-9, which is located in the beta-globin complex of the C57BL/10 mouse, is also reported. Parsimony analysis of these sequences plus two previously reported L1Md sequences allows the determination of an ancestral L1Md sequence. Analysis of the L1Md population indicates that this ancestral sequence is likely to represent a functional L1 sequence. This ancestral sequence confirms that the length (1137 bp and 3900 bp) and relationship (14 bp overlap) of the two large open reading frames previously reported are conserved features of the L1Md family. It also allows the determination of an ancestral amino acid sequence for these two open reading frames. Full-length L1Md elements have one of two sequences tandemly repeated at the 5' end. These two monomers are called A-type and F-type. Our data define the 5' end of A-type full-length L1Md elements. L1Md elements of the A-type have varying numbers of tandemly repeated 208 bp monomers, but each element ends about 78 bp from the 5' end of the terminal 208 bp monomer.