The N-terminal region of RECQL4 lacking the helicase domain is both essential and sufficient for the viability of vertebrate cells: Role of the N-terminal region of RECQL4 in cells

Rothmund-Thomson syndrome (RTS) is a rare genetic disorder characterized by premature aging, developmental abnormalities, and a predisposition to cancer. RTS is caused by mutations in the RECQL4 gene, which encodes one of the five human RecQ helicases. To identify the cellular functions of RECQL4, we generated a chicken DT40 cell line in which RECQL4 expression could be turned off by doxycycline (Dox). Upon exposure to Dox, cells stopped growing and underwent apoptosis. The cells could be rescued by expression of the N-terminal region of RECQL4 (amino acids 1-496), which lacks the helicase domain and has sequence similarity to yeast Sld2, which plays an essential function in the initiation of DNA replication in Saccharomyces cerevisiae. Smaller fragments of the N-terminal region of RECQL4 did not rescue the cells from lethality. RECQL4 gene knockout cells complemented with RECQL4 (1-496) showed relatively high sensitivity to DNA damaging agents that induce double strand breaks and cross-links, suggesting that the C-terminal region including the helicase domain of RECQL4 is involved in the repair of certain types of DNA lesions.     

Sensitivity of RECQL4-deficient fibroblasts from Rothmund–Thomson syndrome patients to genotoxic agents

RECQ helicase protein-like 4 (RECQL4) is a member of the human RECQ family of DNA helicases. Two-thirds of patients with Rothmund–Thomson syndrome (RTS) carry biallelic inactivating mutations in the RECQL4 gene. RTS is an autosomal recessive disorder characterized by poikiloderma, sparse hair, small stature, skeletal abnormalities, cataracts, and an increased risk of cancer. Mutations in two other RECQ helicases, BLM and WRN, are responsible for the cancer predisposition conditions Bloom and Werner syndromes, respectively. Previous studies have shown that BLM and WRN-deficient cells demonstrate increased sensitivity to hydroxyurea (HU), camptothecin (CPT), and 4-nitroquinoline 1-oxide (4NQO). Little is known about the sensitivity of RECQL4-deficient cells to these and other genotoxic agents. The purpose of this study was to determine if RTS cells display any distinct cellular phenotypes in response to DNA damaging agents or replication blocks that could provide insight into the molecular function of the RECQL4 protein. Our results show that primary fibroblasts from RTS patients carrying two deleterious RECQL4 mutations, compared to wild type (WT) fibroblasts, have increased sensitivity to HU, CPT, and doxorubicin (DOX), modest sensitivity to other DNA damaging agents including ultraviolet (UV) irradiation, ionizing radiation (IR), and cisplatin (CDDP), and relative resistance to 4NQO. The RECQ family of DNA helicases has been implicated in the regulation of DNA replication, recombination, and repair. Because HU, CPT, and DOX exert their effects primarily during S phase, these results support a greater role for the RECQL4 protein in DNA replication as opposed to repair of exogenous damage. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Remarkable sequence conservation of the last intron in the PKD1 gene

The last intron of the PKD1 gene (intron 45) was found to have exceptionally high sequence conservation across four mammalian species: human, mouse, rat, and dog. This conservation did not extend to the comparable intron in pufferfish. Pairwise comparisons for intron 45 showed 91% identity (human vs. dog) to 100% identity (mouse vs. rat) for an average for all four species of 94% identity. In contrast, introns 43 and 44 of the PKD1 gene had average pairwise identities of 57% and 54%, and exons 43, 44, and 45 and the coding region of exon 46 had average pairwise identities of 80%, 84%, 82%, and 80%. Intron 45 is 90 to 95 bp in length, with the major region of sequence divergence being in a central 4-bp to 9-bp variable region. RNA secondary structure analysis of intron 45 predicts a branching stem-loop structure in which the central variable region lies in one loop and the putative branch point sequence lies in another loop, suggesting that the intron adopts a specific stem-loop structure that may be important for its removal. Although intron 45 appears to conform to the class of small, G-triplet-containing introns that are spliced by a mechanism utilizing intron definition, its high sequence conservation may be a reflection of constraints imposed by a unique mechanism that coordinates splicing of this last PKD1 intron with polyadenylation.     

Identification of new RECQL4 mutations in Caucasian Rothmund-Thomson patients and analysis of sensitivity to a wide range of genotoxic agents

Rothmund-Thomson syndrome (RTS), a rare recessive autosomal disorder, presents genome instability and clinical heterogeneity with growth deficiency, skin and bone defects, premature aging symptoms and cancer susceptibility. A subset of RTS patients presents mutations of the RECQL4 gene, member of the RecQ family of DNA helicases, including the RECQL2 (BLM) and RECQL3 (WRN) genes, defective in the cancer prone Bloom and Werner syndromes, respectively. Analysis of the RECQL4 gene in six clinically diagnosed RTS patients shows five patients, including two siblings, with eight mutations mainly located in the helicase domain, three patients presenting two mutations. The alterations include four missense mutations, one nonsense mutation and the same frameshift deletion, g.2881delG in exon 9 found in three patients. Seven RECQL4 polymorphisms, two being new, have also been identified. Primary RTS fibroblasts from these RTS patients show no sensitivity to a wide variety of genotoxic agents including ionizing or ultraviolet irradiation, nitrogen mustard, 4NQO, 8-MOP, Cis-Pt, MMC, H2O2, HU, or UV plus caffeine which could be related to the RECQL4 alterations identified here. This is in contrast with the DNA damage sensitive Bloom and Werner cells and highlights the complexity of the numerous RecQ protein functions implicated in the different cellular pathways required for maintaining genomic integrity.     

The N-terminal region of RECQL4 lacking the helicase domain is both essential and sufficient for the viability of vertebrate cells. Role of the N-terminal region of RECQL4 in cells

Rothmund-Thomson syndrome (RTS) is a rare genetic disorder characterized by premature aging, developmental abnormalities, and a predisposition to cancer. RTS is caused by mutations in the RECQL4 gene, which encodes one of the five human RecQ helicases. To identify the cellular functions of RECQL4, we generated a chicken DT40 cell line in which RECQL4 expression could be turned off by doxycycline (Dox). Upon exposure to Dox, cells stopped growing and underwent apoptosis. The cells could be rescued by expression of the N-terminal region of RECQL4 (amino acids 1-496), which lacks the helicase domain and has sequence similarity to yeast Sld2, which plays an essential function in the initiation of DNA replication in Saccharomyces cerevisiae. Smaller fragments of the N-terminal region of RECQL4 did not rescue the cells from lethality. RECQL4 gene knockout cells complemented with RECQL4 (1-496) showed relatively high sensitivity to DNA damaging agents that induce double strand breaks and cross-links, suggesting that the C-terminal region including the helicase domain of RECQL4 is involved in the repair of certain types of DNA lesions.     

The exon/intron organization of the globin gene of Scapharca inaequivalvis homodimeric hemoglobin: unusual intron homology with other bivalve mollusc globin genes

In this study, we have investigated the positions of introns in the globin gene of Scapharca inaequivalvis homodimeric hemoglobin. We found the three exon/two intron organization typical of vertebrate globin genes, with the two introns in highly conserved positions, as it occurs in the A and B globin genes of the tetrameric hemoglobin from the same organism, confirming the absence of the so-called `central intron' found in the globin genes of plants and of some invertebrates. We identified two homodimeric globin genes (3207 and 2723 bp) that differ only with respect to the size of the first intron. Sequence analysis of the two first introns (1668 and 1364 bp) has revealed that they are highly homologous, except for a 569- and 296-bp insertion in each intron I. Interestingly, the two first introns contain regions with an unusually high identity (∼80%) with regions of the first intron of the congeneric clam Anadara trapezia and the related clam Barbatia reveana globin genes, suggesting that these uncoding regions may have played a regulatory role that has subsequently been lost during the course of the evolution.     

Diversification of the rice Waxy gene by insertion of mobile DNA elements into introns.

The waxy (wx) gene of Oryza glaberrima was cloned, and its nucleotide sequence was determined. A waxy mutant of O. glaberrima showing a glutinous phenotype was found to contain a substitution mutation generating a termination codon in the coding region of the wx gene. The Wx sequence of O. glaberrima was different from that of Oryza sativa by substitutions and insertions/deletions, among which only a few substitutions occurred in several exons not to severely alter the amino acid sequence of the Wx protein. The most striking difference observed in introns was a 139-bp deletion (or insertion) in intron 10 of O. glaberrima (or O. sativa). In O. sativa, 125 bp of the 139-bp sequence was flanked by direct repeats of a 14-bp sequence. A sequence homologous to the 125-bp sequence was found in the region preceding exon 2; this sequence was also flanked by direct repeats of another 14-bp sequence. This result and the observation that the 125-bp sequence was interspersed in rice genomes indicate that they are SINEs (short interspersed elements) in the plant system. We also identified a DNA sequence with long terminal inverted repeats in intron 13 of both O. glaberrima and O. sativa. This sequence was present in multiple copies in rice genomes, suggesting that it is a transposable element. These results obtained suggest that mobile DNA elements have diversified the rice Waxy gene by inserting into introns, each of which may originally have a length of about 100 bp.     

Human placental protein 14 gene: sequence and characterization of a short duplication

Differential hybridization of cDNAs corresponding to mRNAs expressed in the human endometrium during the secretory phase or during the first trimester of pregnancy, but not during the proliferative phase, allowed us to isolate and characterize cDNAs encoding human placental protein 14 (PP14). The cDNA was used to isolate the PP14 gene from a human genomic library. The entire gene encompasses 5.05 kb divided into seven exons by six introns. The human PP14 gene shows identical organization with the ovine beta-lactoglobulin gene, as expected from protein homology. Sequencing of 3 kb of the 5'-flanking region of the gene allowed us to characterize a 400-bp duplication of the PP14 gene lying at position -2,660. This duplication was homologous to 100 bp of exon 4 and 300 bp of intron 4, including 180 bp corresponding exactly to the right arm of an Alu element lying on the complementary strand. This homology suggests that this duplication may have arisen through a retroposition event.     

Occurrence of introns in the 16S rRNA genes of members of the genus Thermoproteus

Multiple introns were detected in the 16S rRNA gene of newly isolated Thermoproteus species strains IC-033 and IC-061 and Thermoproteus neutrophilus JCM 9278. In the 16S rRNA gene of strain IC-033, five introns of 627, 762, 636, 33, and 682 bp existed after positions 548, 781, 1092, 1205, and 1213 (according to the Escherichia coli numbering system), respectively. Likewise, strain IC-061 possessed 764-, 32-, and 688-bp introns after positions 781, 1205, and 1213, respectively; and T. neutrophilus JCM 9278 had 34- and 663-bp introns after positions 1205 and 1213, respectively. All the introns carried the putative intron core structures consisting of a bulge-helix-bulge motif and a long stable stem. The large introns carried open reading frames containing the LAGLI-DADG-like motifs in their terminal inserts; however, three out of four large introns of strain IC-033 seemed to incur frameshift mutations. Occurrence of introns at the same insertion sites in the three strains would allow tracing of the evolutionary movements of these introns. Received: 16 February 1998 / Accepted: 27 April 1998     

Cloning and characterization of the mouse short-chain acyl-CoA dehydrogenase gene

Short-chain acyl-CoA dehydrogenase (SCAD) is one of four straight-chain length specific enzymes involved in the first step of fatty acid β-oxidation. To further understand the similarities between the members of this gene family, to characterize how the gene is regulated, and to determine if there is coordinate regulation between these similar genes, we have isolated genomic clones containing the mouse Acads gene. We show that Acads is a compact, single-copy gene approximately 5000 bp in size. We sequenced the entire coding portion of the gene, all of the intron/exon junctions, and an 850-bp segment upstream of the translation start site. We have determined that the gene consists of 10 exons ranging in size from 57 bp to 703 bp, and 9 introns ranging in size from 80 bp to approximately 700 bp. The 5′ region of the mouse Acads gene lacks a TATA box or a CAAT box, is GC rich, and also lacks any similarity to the related gene, medium-chain acyl-CoA dehydrogenase. This is the initial report of the gene structure and 5′ regulatory sequence of the short-chain acyl-CoA dehydrogenase gene in any species. Received: 20 September 1995 / Accepted: 4 December 1995     

Selective constraints on intron evolution in Drosophila

Intron sizes show an asymmetrical distribution in a number of organisms, with a large number of "short" introns clustered around a minimal intron length and a much broader distribution of longer introns. In Drosophila melanogaster, the short intron class is centered around 61 bp. The narrow length distribution suggests that natural selection may play a role in maintaining intron size. A comparison of 15 orthologous introns among species of the D. melanogaster subgroup indicates that, in general, short introns are not under greater DNA sequence or length constraints than long introns. There is a bias toward deletions in all introns (deletion/insertion ratio is 1.66), and the vast majority of indels are of short length (<10 bp). Indels occurring on the internal branches of the phylogenetic tree are significantly longer than those occurring on the terminal branches. These results are consistent with a compensatory model of intron length evolution in which slightly deleterious short deletions are frequently fixed within species by genetic drift, and relatively rare larger insertions that restore intron length are fixed by positive selection. A comparison of paralogous introns shared among duplicated genes suggests that length constraints differ between introns within the same gene. The janusA, janusB, and ocnus genes share two short introns derived from a common ancestor. The first of these introns shows significantly fewer indels than the second intron, although the two introns show a comparable number of substitutions. This indicates that intron-specific selective constraints have been maintained following gene duplication, which preceded the divergence of the D. melanogaster species subgroup.     

Isolation and characterization of a gene encoding cinnamate 4-hydroxylase from <Emphasis Type="Italic">Parthenocissus henryana</Emphasis>

Cinnamate 4-hydroxylase (C4H, EC 1.14.13.11) plays an important role in the phenylpropanoid pathway, which produces many economically important secondary metabolites. A gene coding for C4H, designated as PhC4H (GenBank accession no. DQ211885) was isolated from Parthenocissus henryana. The full-length PhC4H cDNA is 1,747 bp long with a 1,518-bp open reading frame encoding a protein of 505 amino acids, a 40-bp 5′ non-coding region and a 189-bp 3′-untranslated region. Secondary structure of the deduced PhC4H protein consists of 41.78% alpha helix, 15.64% extended strand and 42.57% random coil. The genomic DNA of PhC4H is 2,895 bp long and contains two introns; intron I is 205-bp and intron II is 1,172-bp (GenBank accession no. EU440734). DNA gel blot analysis revealed that there might be a single copy of PhC4H in Parthenocissus henryana genome. By using anchored PCR, a 963-bp promoter sequence was isolated and it contains many responsive elements conserved in the upstream region of PAL, C4H and 4CL including the P-, A-, L- and H-boxes.     

Distributions of exons and introns in the human genome

The human genome is revisited using exon and intron distribution profiles. The 26,564 annotated genes in the human genome (build October, 2003) contain 233,785 exons and 207,344 introns. On average, there are 8.8 exons and 7.8 introns per gene. About 80% of the exons on each chromosome are < 200 bp in length. < 0.01% of the introns are < 20 bp in length and < 10% of introns are more than 11,000 bp in length. These results suggest constraints on the splicing machinery to splice out very long or very short introns and provide insight to optimal intron length selection. Interestingly, the total length in introns and intergenic DNA on each chromosome is significantly correlated to the determined chromosome size with a coefficient of correlation r = 0.95 and r = 0.97, respectively. These results suggest their implication in genome design.     

INTRON REVEALED BY NUCLEOTIDE SEQUENCE OF LARGE SUBUNIT OF RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE FROM CODIUM FRAGILE (CHLOROPHYTA): PHYLOGENETIC ANALYSIS1

The coding sequence for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) from Codium fragile (Suringar) Hariot chloroplast DNA is 1428 bp in length and contains a 1813-bp group II intron. The only other organisms in which introns have been found in the rbcL gene are Euglena and Astasia. The Codium intron likely had a separate origin from the Euglena and Astasia introns, based on comparisons of intron sizes and sequences. Phylogenetic analyses of rbcL nucleotide and amino acid sequences place Codium between Chlorella and two Chlamydomonas spp., indicating that the Chlorophyceae may be polyphyletic.