Barth syndrome: an X-linked cause of fetal cardiomyopathy and stillbirth.

OBJECTIVE: Barth Syndrome (BTHS) is an X-linked multisystem disorder (OMIM 302060) usually diagnosed in infancy and characterized by cardiac problems [dilated cardiomyopathy (DCM) ± endocardial fibroelastosis (EFE) ± left ventricular non-compaction (LVNC)], proximal myopathy, feeding problems, growth retardation, neutropenia, organic aciduria and variable respiratory chain abnormalities. We wished to determine whether BTHS had a significant impact on fetal and perinatal health in a large cohort of family groups originating from a defined region. METHOD: Case note review on 19 families originating from the UK and known to the Barth Syndrome Service of the Bristol Royal Hospital for Children. RESULTS: Details are presented on six kindreds (32%) with genetically and biochemically proven BTHS that demonstrate a wider phenotype including male fetal loss, stillbirth and severe neonatal illness or death. In these families, 9 males were stillborn and 14 died as neonates or infants but there were no losses of females. BTHS was definitively proven in five males with fetal onset of DCM ± hydrops/EFE/LVNC. CONCLUSION: These findings stress the importance of considering BTHS in the differential diagnosis of unexplained male hydrops, DCM, EFE, LVNC or pregnancy loss, as well as in neonates with hypoglycemia, lactic acidosis and idiopathic mitochondrial disease.

Molecular analysis of a constitutional X-autosome translocation in a female with muscular dystrophy.

The gene responsible for Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) maps to the X chromosome short arm, band Xp21. In a few females with DMD or BMD, the Xp21 region is disrupted by an X-autosome translocation. Accumulating evidence suggests that the exchange has physically disrupted the DMD/BMD locus to cause the disease. One affected female with a t(X;21)(p21;p12) translocation was studied in detail. The exchange points from both translocation chromosomes were cloned, restriction-mapped, and sequenced. The translocation is reciprocal, but not conservative. A small amount of DNA is missing from the translocated chromosomes; 71 to 72 base pairs from the X chromosome and 16 to 23 base pairs from the 28S ribosomal gene on chromosome 21.

Sequence and structure correlation of human ribosomal transcribed spacers.

We report the sequences of the transcribed spacers of human rRNA that now allow us to piece together the entire primary transcript sequence of approximately 13.3 x 10(3) base-pairs. Comparison of transcribed spacer sequences with those of variable regions of rRNA and with those of the non-transcribed spacers supports the hypothesis that the variable regions are descended from transcribed spacers. Nucleotide sequence-derived secondary structures for the 5' external transcribed spacer and for internal transcribed spacers 1 and 2 match both the sizes and shapes of the structures that were visualized 15 years ago on electron micrographs. Parts of these structures are conserved in mammals and may be related to transcript processing.

Studies of the inheritance of human ribosomal DNA variants detected in two-dimensional separations of genomic restriction fragments.

We have investigated the variation in human ribosomal DNA repeat units as revealed in two-dimensional electrophoretic separates of genomic restriction fragments that were end-labeled at NotI cleavage sites. The transcribed portion of the ribosomal DNA results in approximately 20 labeled fragments visible on each gel as multicopy spots. We have mapped these spots to the sequences responsible for their appearance on the gels, based on their migration positions and direct sequencing of spots, and describe several previously unreported sources of variation. By studying mother/father/child families we gained information on how much of the between-repeats variation is due to differences between and within repeat arrays on homologous chromosomes. Two instances in which a child exhibited more copies of a particular fragment than were present in the parents are described and hypothesized to be due to events such as multiple unequal sister-chromatid exchanges or gene conversions.

Vasoactive intestinal peptide receptor expression in chronic periapical lesions.

AIM: To use radioreceptor analysis for evaluating whether vasoactive intestinal peptide (VIP) receptors are present in chronic periapical lesions and to determine whether differences in its expression are found according to the size of the lesions. METHODOLOGY: Twelve periapical lesions were obtained from teeth diagnosed with chronic apical periodontitis and indicated for endodontic surgery; they were classified according to the size of the lesion in two groups of six samples (lesion size greater or smaller than 5 mm), and then processed and labelled with (125)I-VIP. Binding sites were identified by (125)I-VIP and standard VIP competition assays. Mann-Whitney's test was used to establish statistically significant differences in the VIP receptor expression between groups. RESULTS: Vasoactive intestinal peptide receptor expression was found in all periapical lesion samples. There was a statistically significantly higher expression in periapical lesions <5 mm (P < 0.001). CONCLUSION: Vasoactive intestinal peptide receptors were expressed in chronic periapical lesions with levels inversely proportional to lesion size.

Complete sequence of the 43-kb human ribosomal DNA repeat: analysis of the intergenic spacer.

We have sequenced the remaining 12.4 kb of the 30-kb human ribosomal DNA intergenic spacer (IGS), which allows us to piece together both a complete IGS and a 43-kb rDNA unit. The sequence of the complete IGS reveals a collection of sequence motifs that can be correlated with functions known or expected to reside in the rDNA repeat: modulation of transcription, recombination, initiation of DNA replication, and chromosomal organization. Finally, we find that IGS accumulates variation at a much higher rate than the transcribed regions. This finding leads us to correlate sequence character with types of mutation and sequence context with rate of mutation.

Beyond ribosomal DNA: on towards the telomere.

We have sequenced and analyzed 8.3 kb of sequence adjacent and distal to the human ribosomal DNA (rDNA); this distal sequence connects to the rDNA cluster just 4 kb upstream of the first promoter and is shared among the acrocentric chromosomes and, at least in part, it is also present in other primates. The sequence differs in character from that of the rDNA intergenic spacer (IGS) in that it does not contain long stretches of either polypyrimidine or polypurine. However, just like the IGS, it contains numerous repetitive elements, including retroposed fragments of 28S rRNA and large pieces of the IGS. In addition, we show that the rDNA clusters are not interrupted by other sequences and do not recombine with this distal segment.

Incognito rRNA and rDNA in databases and libraries.

Both ribosomal DNA (rDNA) and ribosomal RNA (rRNA) are over-represented in the starting material for genomic and cDNA libraries; thus, their sequences have the potential of repeatedly entering the various databases. When DNA (both transcribed and intergenic spacer regions) is used as query sequence, a great number of matches are found in the databases, particularly in the EST database, and to a lesser extent among genomic sequences and STSs, which are not identified as rDNA. We discuss the following explanations for the widespread occurrence of rDNA in cDNA and genomic DNA libraries: pseudogenes of rRNA in other genomic locations, mRNA-derived pseudogenes that reside in rDNA, cDNAs derived from rRNA [either by self-priming or by internal oligo(dT) priming], cDNAs derived from actual transcripts of the rDNA intergenic spacer, and genomic DNA contamination of RNA preparations. Because so many database entries contain unidentified rDNA, we recommend that all sequence submissions be checked (by the submitters) for the presence of structural RNAs in addition to repetitive sequences.

Human rDNA: evolutionary patterns within the genes and tandem arrays derived from multiple chromosomes.

Human rDNA forms arrays on five chromosome pairs and is homogenized by concerted evolution through recombination and gene conversion (loci RNR1, RNR2, RNR3, RNR4, RNR5, OMIM: 180450). Homogenization is not perfect, however, so that it becomes possible to study its efficiency within genes, within arrays, and between arrays by measuring and comparing DNA sequence variation. Previous studies with randomly cloned genomic DNA fragments showed that different parts of the gene evolve at different rates but did not allow comparison of rDNA sequences derived from specific chromosomes. We have now cloned and sequenced rDNA fragments from specific acrocentric chromosomes to (1) study homogenization along the rDNA and (2) compare homogenization within chromosomes and between homologous and nonhomologous chromosomes. Our results show high homogeneity among regulatory and coding regions of rDNA on all chromosomes, a surprising homogeneity among adjacent distal non-rDNA sequences, and the existence of one to three very divergent intergenic spacer classes within each array.

Mutations in the L-arabinose operon of Escherichia coli B/r with reduced initiator function.

Partial reversion mutants derived from a strain containing a strongly polar initiator-defective mutation (araI1036) in the L-arabinose operon were found to have several characteristics expected of mutants with reduced initiator function. These reversion mutations are cotransduced with the ara region and are probably within the araI region. Furthermore, they permit induction of the L-arabinose operon to a level only one-third of the normal wild-type level. These partially functional initiator regions reduce the expression of structural genes in the cis position only; they function quite independently of wild-type or defective initiator regions in the trans position. These mutants exhibit a two- to threefold increase in the rate of expression of ara operon genes within one-tenth of a generation after a shift of the growth temperature from 28 to 42 degrees C. This suggests that the temperature optimum for initiation of operon expression is higher for the partial revertant strains than it is for strains containing a wild-type initiator region.

The human 18S ribosomal RNA gene: evolution and stability.

We report the 1,870-base-pair primary sequence of a human 18S rRNA gene and propose a secondary structure based on this sequence and the general mammalian structure. A basic secondary structure for the small subunit rRNA has been preserved throughout evolution by compensatory and neutral base changes in double-stranded regions. The molecule contains eight regions that can vary in structure and that comprise 432 bases, while 1,438 bases belong to regions of conserved structure among all species tested. The conserved regions show a remarkably low sequence divergence rate of 0.1% between the human and mouse genes over the approximately 80 million years since the mammalian radiation. This value may make the small subunit rDNA the most highly conserved sequence known. Sequence conservation in higher eukaryotes with multiple copies of the gene is probably achieved by the combination of strong selection and the correction of tandem genes by unequal homologous exchange.

Independent insertion of Alu elements in the human ribosomal spacer and their concerted evolution.

A 2,700-bp segment of human ribosomal DNA (rDNA) spacer upstream of the rRNA promoter contains a set of four Alu elements, two in the direction of rRNA transcription and two in the opposite orientation. We report and compare the sequences of these Alu elements found in three rDNA clones and seek to determine the origin of the cluster, either from a single insertion followed by duplications or from multiple simultaneous or independent insertions. The high (20%-27%) divergence among members of a set and the lack of similarity/complementarity of sequences flanking different members of the set demonstrate the independent insertion of each of the four Alu elements into A-rich sequences on the appropriate strand of the rDNA. We also demonstrate that the Alu sets found in different rDNA repeats are subject to concerted evolution, yielding divergences of only 0.4%-3% between Alu elements in equivalent positions. However, the pairs of adjacent similarly oriented Alu elements do not show reduced divergence, indicating that there is no recombination or gene conversion between similarly oriented but not equivalently positioned Alu elements. Finally, crossing-over must occur in the rDNA junction region between Alu element 3 and the nonribosomal sequences at the telomere end of the acrocentric chromosome, so that the Alu elements of the terminal rDNA repeats and the terminal repeats themselves evolve in concert with the rDNA repeats located internally in the tandem array.

Human 28S ribosomal RNA sequence heterogeneity.

DNA sequencing of several cloned human 28S ribosomal RNA gene fragments has revealed sequence heterogeneity (1) but it was not clear whether these are inactive pseudogenes or are active genes that are transcribed and represented in ribosomes. S1 nuclease analysis allowed us to examine the population of ribosomal RNA molecules of a cell, and we found that 28S rRNA is a heterogeneous assortment of molecules in both mono- and polysomal preparations. Sequence variation, although largely concentrated in variable regions of the molecule, apparently also occurs in the conserved regions.

The secondary structure of human 28S rRNA: the structure and evolution of a mosaic rRNA gene.

We have determined the secondary structure of the human 28S rRNA molecule based on comparative analysis of available eukaryotic cytoplasmic and prokaryotic large-rRNA gene sequences. Examination of large-rRNA sequences of both distantly and closely related species has enabled us to derive a structure that accounts both for highly conserved sequence tracts and for previously unanalyzed variable-sequence tracts that account for the evolutionary differences in size among the large rRNAs. Human 28S rRNA is composed of two different types of sequence tracts: conserved and variable. They differ in composition, degree of conservation, and evolution. The conserved regions demonstrate a striking constancy of size and sequence. We have confirmed that the conserved regions of large-rRNA molecules are capable of forming structures that are superimposable on one another. The variable regions contain the sequences responsible for the 83% increase in size of the human large-rRNA molecule over that of Escherichia coli. Their locations in the gene are maintained during evolution. They are G + C rich and largely nonhomologous, contain simple repetitive sequences, appear to evolve by frequent recombinational events, and are capable of forming large, stable hairpins. The secondary-structure model presented here is in close agreement with existing prokaryotic 23S rRNA secondary-structure models. The introduction of this model helps resolve differences between previously proposed prokaryotic and eukaryotic large-rRNA secondary-structure models.

Fixation times of retroposons in the ribosomal DNA spacer of human and other primates.

We have investigated the presence/absence of two types of retroposed sequences found in human ribosomal DNA in equivalent positions in chimpanzee, gorilla, orangutan, gibbon, and rhesus monkey rDNA. These sequences are one pseudogene derived from the single-copy cdc27hs gene and seven complete Alu elements. The 2-kb pseudogene is present in the apes but not in Old World monkeys, indicating fixation in an ape ancestor. Five of the Alu elements are shared by the whole set of primates studied, indicating insertion and fixation prior to the split of the ape and Old World monkey lineages. One is absent only from the rhesus monkey rDNA, and another is absent from both gibbon and rhesus rDNA, indicating fixation at different times in primate evolutionary history. Since branching times for the primate phylogenetic tree are known from a combination of the fossil record and multiple molecular data sets, it is possible to compare Alu fixation times determined from the phylogenetic information with those calculated from Alu element mutation rates.

Human ribosomal RNA variants from a single individual and their expression in different tissues.

We have investigated the extent of sequence variation in human ribosomal RNA (rRNA) genes and the expression of specific rRNA gene variants in different tissues of an individual. Focusing on the fifth variable region (V5; nt 2065-2244) of the 28S rRNA gene, we find that sequence differences between rRNA genes of a single individual are characterized by differences in number of repeats of simple sequences at four specific sites. These data support and extend previous findings which show similar V5 sequence variation in rRNA genes from a group of individuals. We performed experiments to determine if there is differential gene expression within the rRNA multigene family. From the analysis of data of six variant V5 probes protected from RNase digestion by rRNAs isolated from different tissues of the individual, we conclude that each variant rRNA is present in a similar proportion in these tissues, whereas the actual contributions of variants differ, their relative proportion is maintained from tissue to tissue in an individual. We favor the explanation of a gene dosage effect over that of a regulated gene effect to account for this pattern of rRNA gene expression. In addition, computer generated secondary structure models of each V5 clone structure predict the same three helix structure with the regions of sequence variation contained in one stem-loop structure.

Ribosomal RNA gene sequences and hominoid phylogeny.

Sequences totaling 3,500 bases from the 28S rRNA gene and from one of the ribosomal internal transcribed spacers (ITS1) have been determined for human, chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), and orangutan (Pongo pygmaeus). Analyses of the rRNA alignments show (1) a clustering of substitutions in the "variable regions" of the 28S gene, (2) a 1.5-3-fold increase in divergence in the transcribed spacer over that in the exon, and (3) that human and chimpanzee are the most closely related pair, in agreement with the results of Miyamoto et al., Sibley and Ahlquist, and Caccone and Powell.

Definition of a second dimeric subfamily of human alpha satellite DNA.

We describe a new human subfamily of alpha satellite DNA. The restriction endonuclease XbaI cleaves this subfamily into a collection of fragments which are heterogeneous with respect to size. We compared the sequences of 6 clones from four different XbaI size classes. Clones from a single size class were not necessarily more related than clones from different classes. Clones from different size classes were found to produce almost identical hybridization patterns with XbaI-digested human genomic DNA. All clones were found to share a common dimeric repeat organization, with dimers exhibiting about 84% sequence identities, indicating that the clones evolved from a common progenitor alphoid dimer. We show that this subfamily, and the EcoRI dimer subfamily originally described by Wu and Manuelidis, evolved from different progenitor alphoid dimers, and therefore represent distinct human alphoid subfamilies.

Complementarity between ferritin H mRNA and 28 S ribosomal RNA.

We have found an interesting complementarity in sequences of human ferritin H mRNA and 28 S ribosomal RNA. Immediately upstream of the initiating AUG in the ferritin mRNA is a stretch of 67 nucleotides which contains sequences complementary to several regions in 28 S RNA. One such region can form 55 base pairings with the 5' noncoding region of the ferritin H mRNA. Most of the complementarity is due to repeats of CCG in the ferritin mRNA and GGC in the ribosomal RNA. The regions of complementarity in the 28 S RNA appear to be expansion sequences that have arisen in the evolution of eukaryotic ribosomal RNA. We suggest that interaction of ferritin mRNA and 28 S RNA may function to regulate the stability and/or translatability of ferritin mRNA.