Homology-dependent inactivation of LTR retrotransposons in genomes of Aspergillus fumigatus and A. nidulans

Repeat-induced point mutation (RIP) is the most intriguing among the known mechanisms of repeated sequences inactivation because of its ability to produce irreversible mutation of repeated DNA. Discovered for the first time in Neurospora crassa, RIP is characterized by C:G to T:A transitions in duplicated sequences. The mechanisms and distribution of RIP are still purely investigated. Mobile elements are a common target for the processes which lead to homology-dependent silencing because of their ability to propagate themselves. We have done comparative analysis of LTR retrotransposons in genomic scale from genomes of two aspergilli fungi--Aspergillus funmigatus and A. nidulans, based on several copies we reconstructed "de-RIP" retroelements. Investigations of frequencies of CpG, CpA and TpG sites, which are potential targets for mutagenesis, showed the much lower frequencies of these sites in mobile elements in comparison with structural genes. LTR retrotransposons from A. fumigatus and A. nidulans have different ratio of types of substitutions. Our analysis indicates that two investigated fungi have or had the RIP-like processes for repeated sequences inactivation, in various modes. Whereas in A. fumigatus the context for mutagenesis consists of both CpG and CpA sites, in A. nidulans inactivation seems to proceed only on CpG dinucleotides. The present investigation gives a theoretical background for planning of experimental studying of RIP inactivation in aspergilli.     

Structure of the human ornithine transcarbamylase gene

Complementary and genomic DNA clones corresponding to the human ornithine transcarbamylase (OTC) [EC 2.1.3.3]mRNA have been isolated and analyzed. The OTC gene is about 73 kilobase pairs (kb) long and contains 10 exons interrupted by 9 introns of highly variable sizes. The smallest intron is 80 base pairs and the largest, 21.7 kb. The 5'- and 3'-flanking regions, entire exons and all the exon/intron boundaries were sequenced. The nucleotide and deduced amino acid sequences of isolated OTC cDNAs as well as the corresponding regions of the genomic DNA were compared with those of human OTC cDNA (Horwich, A.L., Fenton, W.A., Williams, K.R., Kalousek, F., Kraus, J.P., Doolittle, R.F., Koningsberg, W., & Rosenberg, L.E. (1984) Science 224, 1068-1074). We found 20 nucleotide substitutions among these sequences, of which 6 related to amino acid changes. The nature of these nucleotide substitutions is discussed.     

Collateral damage: Spread of repeat-induced point mutation from a duplicated DNA sequence into an adjoining single-copy gene inNeurospora crassa

Repeat-induced point mutation (RIP) is an unusual genome defense mechanism that was discovered inNeurospora crassa. RIP occurs during a sexual cross and induces numerous G : C to A : T mutations in duplicated DNA sequences and also methylates many of the remaining cytosine residues. We measured the susceptibility of theerg-3 gene, present in single copy, to the spread of RIP from duplications of adjoining sequences. Genomic segments of defined length (1, 1.5 or 2 kb) and located at defined distances (0, 0.5, 1 or 2 kb) upstream or downstream of theerg-3 open reading frame (ORF) were amplified by polymerase chain reaction (PCR), and the duplications were created by transformation of the amplified DNA. Crosses were made with the duplication strains and the frequency oferg-3 mutant progeny provided a measure of the spread of RIP from the duplicated segments into theerg-3 gene. Our results suggest that ordinarily RIP-spread does not occur. However, occasionally the mechanism that confines RIP to the duplicated segment seems to fail (frequency 0.1–0.8%) and then RIP can spread across as much as 1 kb of unduplicated DNA. Additionally, the bacterialhph gene appeared to be very susceptible to the spread of RIP-associated cytosine methylation.     

Integration host factor binds to a unique class of complex repetitive extragenic DNA sequences in Escherichia coli

Interspersed repeated DNA sequences are characteristic features of both prokaryotic and eukaryotic genomes. REP sequences are defined as conserved repetitive extragenic palindromic sequences and are found in Escherichia coli, Salmonella typhimurium and other closely related enteric bacteria. These REP sequences may participate in the folding of the bacterial chromosome. In this work we describe a unique class of 28 conserved complex REP clusters, about 100bp long, in which two inverted REPs are separated by a singular integration host factor (IHF) recognition sequence. We term these sequences RIP (for repetitive IHF-binding palindromic) elements and demonstrate that IHF binds to them specifically. It is estimated that there are about 70 RIP elements in E. coli. Our analysis shows that the RIP elements are evenly distributed around the bacterial chromosome. The possible function of the RIP element is discussed.     

Comparative study of the genomic organization of DNA repeats within the 5′-flanking region of the natural resistance-associated macrophage protein gene (NRAMP1) between humans and great apes

The human NRAMP1 gene located on Chromosome (Chr) region 2q35 is a candidate gene for increased risk of infection by several intracellular macrophage parasites, including M. tuberculosis and M. leprae. In search for a possible mutational hot spot, we have analyzed a 3.5-kb region 5′ to NRAMP1 that is highly enriched for DNA repeat sequences. The repeat sequences could be grouped into one Mer element and six Alu elements, representing five Alu subfamilies, that had integrated in the same DNA region during successive rounds of Alu retropositional activity. Comparative sequence analysis of the Alu cluster region in humans, chimpanzee (Pan paniscus), and gorilla (Gorilla gorilla) revealed only modest sequence variability and failed to detect any evidence for genomic instability of the highly repetitive DNA region. These results show that sequence length variants in the Alu-flanking regions as well as nucleotide substitutions are the most common genomic variations even in a region of extreme Alu-clustering. Moreover, the high degree of sequence conservation among three primate species argues against the Alu cluster being the site of frequent genomic rearrangements or other frequent genetic events that might influence NRAMP1 expression. Received: 20 September 1997 / Accepted: 23 January 1998     

Human glyceraldehyde-3-phosphate dehydrogenase pseudogenes: Molecular evolution and a possible mechanism for amplification

We screened two human genomic libraries and isolated 14 different clones, designated λG1 and EG1-EG13, homologous to human glyceraldehyde-3-phosphate dehydrogenase (GAPD) cDNA. Subcloning and sequencing these recombinant phages led us to classify them as five different pseudogenes (ψG1–ψG5). All these sequences show such features typical of processed pseudogenes as numerous mutations, insertions, and deletions. The identity of numerous mutated sites among these pseudogenes and the presence of two Alu sequences flanking both ends of ψG1 suggest that GAPD pseudogenes originated from a unique reverse transcribed mRNA followed by gene duplication. The rate of nucleotide substitutions per site per year for known GAPD functional genes is low both for the synonymous substitutions (1.87×10⁻⁹) and for the nonsynonymous substitutions (0.12￠10⁻⁹) and indicates that the GAPD cDNA sequence is well conserved not only at the amino acid level, but also at the nucleotide level. The rate of nucleotide substitutions per site per year for GAPD pseudogenes shows a higher value (5.9×10⁻⁹) and suggests that these pseudogenes do not have any functional role. This work was supported by grants from the Consiglio Nazionale delle Ricerche and the Ministero Pubblica Istruzione (Rome, Italy). Special acknowledgment is given to the “Progetto Finalizzato Ingegneria Genetica e Basi Molecolari delle Malattie Ereditarie.”     

Implications of the Plastid Genome Sequence of Typha (Typhaceae, Poales) for Understanding Genome Evolution in Poaceae

Plastid genomes of the grasses (Poaceae) are unusual in their organization and rates of sequence evolution. There has been a recent surge in the availability of grass plastid genome sequences, but a comprehensive comparative analysis of genome evolution has not been performed that includes any related families in the Poales. We report on the plastid genome of Typha latifolia, the first non-grass Poales sequenced to date, and we present comparisons of genome organization and sequence evolution within Poales. Our results confirm that grass plastid genomes exhibit acceleration in both genomic rearrangements and nucleotide substitutions. Poaceae have multiple structural rearrangements, including three inversions, three genes losses (accD, ycf1, ycf2), intron losses in two genes (clpP, rpoC1), and expansion of the inverted repeat (IR) into both large and small single-copy regions. These rearrangements are restricted to the Poaceae, and IR expansion into the small single-copy region correlates with the phylogeny of the family. Comparisons of 73 protein-coding genes for 47 angiosperms including nine Poaceae genera confirm that the branch leading to Poaceae has significantly accelerated rates of change relative to other monocots and angiosperms. Furthermore, rates of sequence evolution within grasses are lower, indicating a deceleration during diversification of the family. Overall there is a strong correlation between accelerated rates of genomic rearrangements and nucleotide substitutions in Poaceae, a phenomenon that has been noted recently throughout angiosperms. The cause of the correlation is unknown, but faulty DNA repair has been suggested in other systems including bacterial and animal mitochondrial genomes.     

Expression and Characterization of Human Tissue Kallikrein Variants

Human tissue kallikrein is a serine protease implicated in the pathology of various inflammatory disorders. As one of the two principal enzymes that generate proinflammatory kinin peptidesin vivo,tissue kallikrein represents an attractive target for therapeutic intervention in diseases such as asthma, pancreatitis, and rheumatoid arthritis. Three distinct human tissue kallikrein variants, differing in one or two amino acid substitutions, are predicted to exist based on genomic or cDNA nucleotide sequences derived from different tissues. The effects of these substitutions on the biochemical properties of tissue kallikrein are unknown but could, in principle, confer tissue-specific functions on the enzyme or affect the clinical utility of specific kallikrein inhibitors. All three variants, as well as a deglycosylated derivative, were expressed in high yield as recombinant proteins inPichia pastoris.The recombinant kallikrein variants and natural urinary kallikrein all hydrolyzed synthetic peptides with similar specificity and efficiency and released kallidin from kininogen at comparable rates. Similarly, no significant differences were observed in the interactions between kallikrein variants and protein inhibitors such as SBTI, α₁-PI, and aprotinin. We conclude that the known tissue kallikrein variants represent allelic variants and are not likely to have tissue-specific activity related to the amino acid substitutions.     

Isolation and characterization of the CRIPTO autosomal gene and its X-linked related sequence.

We have previously reported on the identification of a cDNA clone encoding a novel human growth factor, named "CRIPTO," that is abundantly expressed in undifferentiated human NTERA-2 clone D1 (NT2/D1) and mouse (F9) teratocarcinoma cells. We now report the organization and nucleotide sequence of two related genomic sequences. One (CR-1) corresponds to the structural gene encoding the human CRIPTO protein expressed in the undifferentiated human teratocarcinoma cells, and the other (CR-3) corresponds to a complete copy of the mRNA containing seven base substitutions in the coding region representing both silent and replacement substitutions. The 440 bp 5' to the CAP site of CR-1 are preserved in CR-3. CR-1 maps to chromosome 3, and CR-3 maps to Xq21-q22. Southern blot analysis reveals that multiple CRIPTO-related DNA sequences are present in the human as well as in the mouse genome.     

Distinct evolutionary patterns of Oryza glaberrima deciphered by genome sequencing and comparative analysis

Here we present the genomic sequence of the African cultivated rice, Oryza glaberrima, and compare these data with the genome sequence of Asian cultivated rice, Oryza sativa. We obtained gene‐enriched sequences of O. glaberrima that correspond to about 25% of the gene regions of the O. sativa (japonica) genome by methylation filtration and subtractive hybridization of repetitive sequences. While patterns of amino acid changes did not differ between the two species in terms of the biochemical properties, genes of O. glaberrima generally showed a larger synonymous–nonsynonymous substitution ratio, suggesting that O. glaberrima has undergone a genome‐wide relaxation of purifying selection. We further investigated nucleotide substitutions around splice sites and found that eight genes of O. sativa experienced changes at splice sites after the divergence from O. glaberrima. These changes produced novel introns that partially truncated functional domains, suggesting that these newly emerged introns affect gene function. We also identified 2451 simple sequence repeats (SSRs) from the genomes of O. glaberrima and O. sativa. Although tri‐nucleotide repeats were most common among the SSRs and were overrepresented in the protein‐coding sequences, we found that selection against indels of tri‐nucleotide repeats was relatively weak in both African and Asian rice. Our genome‐wide sequencing of O. glaberrima and in‐depth analyses provide rice researchers not only with useful genomic resources for future breeding but also with new insights into the genomic evolution of the African and Asian rice species.     

Organization, polymorphism, and expression of the human T-cell receptor AV1 subfamily

 At least 32 mostly single-member subfamilies of T-cell receptor alpha variable (TCRAV) genes have been described in humans. The AV1 subfamily is the largest, estimated by hybridization to contain as many as five members. However, a search of nucleotide sequence databases reveals a much greater number of unique sequences corresponding to this subfamily. In order to resolve this discrepancy between hybridization and nucleotide sequencing data, and to better understand the nature of variability among variable genes within a large subfamily, a genomic characterization of the AV1 subfamily in humans was carried out. Total genomic DNA, as well as isolated genomic clones spanning the TCRA region were screened for members of the AV1 subfamily by polymerase chain reaction (PCR) and nucleotide sequencing as well as by hybridization. A total of eight AV1 genes were identified and their nucleotide sequences were determined. Three of the sequences represent new genes. Based on structural features and the results of PCR screening of cDNA, none of these new genes appear to be functional. Several additional previously reported AV1 sequences were determined to represent alleles of AV1 genes, and simple PCR restriction digest assays were established for their detection. Use of each of the identified AV1 genes as hybridization probes failed to reveal any additional hybridizing bands. Thus the AV1genes represent the largest TCRAV subfamily with a maximum of eight members, several of which have common allelic forms. Received: 7 November 1996 / Revised: 5 December 1996     

Rare genomic changes as a tool for phylogenetics

DNA sequence data have offered valuable insights into the relationships between living organisms. However, most phylogenetic analyses of DNA sequences rely primarily on single nucleotide substitutions, which might not be perfect phylogenetic markers. Rare genomic changes (RGCs), such as intron indels, retroposon integrations, signature sequences, mitochondrial and chloroplast gene order changes, gene duplications and genetic code changes, provide a suite of complementary markers with enormous potential for molecular systematics. Recent exploitation of RGCs has already started to yield exciting phylogenetic information.     

Determination of the gene structure of human oligophrenin-1 and identification of three novel polymorphisms by screening of DNA from 164 patients with non-specific X-linked mental retardation

We have recently shown that mutations in oligophrenin-1 (OPHN1) are responsible for non-specific X-linked mental retardation (MRX). The structure of the gene encoding the OPHN1 protein was determined by isolation of genomic DNA clones from the human cosmid library. Genomic fragments containing exons were sequenced, and the sequences of the exons and flanking introns were defined. Knowledge of the genomic structure of the OPHN1 gene, which spans at least 500 kb and consists of 25 exons, will facilitate the search for additional mutations in OPHN1. OPHN1 was screened for mutations in 164 subjects with non-specific mental retardation. Three nucleotide substitutions were identified, one of which was a silent mutation in the codon threonine 301 at position 903 (G-->C). The other substitutions were located in exon 2, a G-->A substitution at position 133 (A45T), and in exon 10, a C-->T substitution at position 902 (T301M), but these are common polymorphisms rather than disease-causing mutations.     

The nucleotide sequence of the murine I-E beta b immune response gene: evidence for gene conversion events in class II genes of the major histocompatibility complex.

We have determined the DNA sequence of the murine I-E beta b immune response gene of the major histocompatibility complex (MHC) of the C57BL/10 mouse and compared it with the sequence of allelic I-E and non-allelic I-A genes from the d and k haplotypes. The polymorphic exon sequences which encode the first extracellular globular domain of the E beta domain show approximately 8% nucleotide substitutions between the E beta b and E beta d alleles compared with only approximately 2% substitutions for the intron sequences. This suggests that an active mechanism such as micro gene conversion events drive the accumulation of these mutations in the polymorphic exons. The fact that several of the nucleotide changes are clustered supports this hypothesis. The E beta b and E beta k genes show approximately 2-fold fewer nucleotide substitutions than the E beta d/E beta b pair. The A beta bm12, a mutant I-A beta b gene from the C57BL/6 mouse, has been shown to result from three nucleotide changes clustered in a short region of the beta 1 domain, which suggests that a micro gene conversion event caused this mutation. We show here that the E beta b gene is identical to the non-allelic A beta bm12 DNA sequence in the mutated region and suggest, therefore, that the E beta b gene was the donor sequence for this intergenic transfer of genetic information. Diversity in class II MHC genes appears therefore to be generated, at least in part, by the same mechanism proposed for class I genes: intergenic transfer of short DNA regions between non-allelic genes.     

The analysis of nucleotide substitutions, gaps, and recombination events between RHD and RHCE genes through complete sequencing

We determined the entire nucleotide sequences of all introns within the RHD and RHCE genes by amplifying genomic DNA using long PCR methods. The RHD and RHCE genes were 57,295 and 57,831 bp in length, respectively. Aligning both genes revealed 138 gaps (insertions and deletions) below 100 bp, 1116 substitutions in all introns and all exons (coding region), and 5 gaps of over 100 bp. Homologies (%) between the RH genes were 93.8% over all introns and coding exons and 91.7% over all exons and introns. Various short tandem repeats (STRs) and many interspersed nuclear elements were identified in both genes. The proportions of Alu sequences in the RHD and RHCE genes were 25.9 and 25.7%, respectively and these Alu sequences were concentrated in several regions. We confirmed multiple recombinations in introns 1 and 2. Such multiple recombination, which probably arose due to the concentrations of Alu sequences and the high level of the homology (%), is one of most important factors in the formation and evolution of RH gene. The variability of the Rh system may be generated because of these features of RH genes. Apparent mutational hotspots and regions with low of K values (the numbers of substitutions per nucleotide site) caused by recombinations as well as true mutational hotspots may be found in human genome. Accordingly, in searching for and identifying single nucleotide polymorphisms (SNPs) especially in noncoding regions, apparent mutational hotspots and areas of low K values by recombination should be noted since the unequal distribution of SNPs will reduce the power of SNPs as genetic maker. Combining the complete sequences' data of both RH genes with serological findings will provide beneficial information with which to elucidate the mechanism of recombination, mutation, polymorphism, and evolution of other genes containing the RH gene as well as to analyze Rh variants and develop new methods of Rh genotyping.     

Recombination-Independent Recognition of DNA Homology for Repeat-Induced Point Mutation (RIP) Is Modulated by the Underlying Nucleotide Sequence

Haploid germline nuclei of many filamentous fungi have the capacity to detect homologous nucleotide sequences present on the same or different chromosomes. Once recognized, such sequences can undergo cytosine methylation or cytosine-to-thymine mutation specifically over the extent of shared homology. In Neurospora crassa this process is known as Repeat-Induced Point mutation (RIP). Previously, we showed that RIP did not require MEI-3, the only RecA homolog in Neurospora, and that it could detect homologous trinucleotides interspersed with a matching periodicity of 11 or 12 base-pairs along participating chromosomal segments. This pattern was consistent with a mechanism of homology recognition that involved direct interactions between co-aligned double-stranded (ds) DNA molecules, where sequence-specific dsDNA/dsDNA contacts could be established using no more than one triplet per turn. In the present study we have further explored the DNA sequence requirements for RIP. In our previous work, interspersed homologies were always examined in the context of a relatively long adjoining region of perfect homology. Using a new repeat system lacking this strong interaction, we now show that interspersed homologies with overall sequence identity of only 36% can be efficiently detected by RIP in the absence of any perfect homology. Furthermore, in this new system, where the total amount of homology is near the critical threshold required for RIP, the nucleotide composition of participating DNA molecules is identified as an important factor. Our results specifically pinpoint the triplet 5''-GAC-3'' as a particularly efficient unit of homology recognition. Finally, we present experimental evidence that the process of homology sensing can be uncoupled from the downstream mutation. Taken together, our results advance the notion that sequence information can be compared directly between double-stranded DNA molecules during RIP and, potentially, in other processes where homologous pairing of intact DNA molecules is observed.