Estrogen receptor α regulates area-adjusted bone mineral content in late pubertal girls

Context: Whether the action of estrogen in skeletal development depends on estrogen receptor α as encoded by the ESR1 gene is unknown. Objectives: The aim of this study was to establish whether the gain in area-adjusted bone mineral content (ABMC) in girls occurs in late puberty and to examine whether the magnitude of this gain is related to ESR1 polymorphisms. Design: We conducted a cross-sectional analysis. Setting: The study involved the Avon Longitudinal Study of Parents and Children (ALSPAC), a population-based prospective study. Participants: Participants included 3097 11-yr-olds with DNA samples, dual x-ray absorptiometry measurements, and pubertal stage information. Outcomes: Outcome measures included separate prespecified analyses in boys and girls of the relationship between ABMC derived from total body dual x-ray absorptiometry scans and Tanner stage and of the interaction between ABMC, Tanner stage, and ESR1 polymorphisms. Results: Total body less head and spinal ABMC were higher in girls in Tanner stages 4 and 5, compared with those in Tanner stages 1, 2, and 3. In contrast, height increased throughout puberty. No differences were observed in ABMC according to Tanner stage in boys. For rs2234693 (PvuII) and rs9340799 (XbaI) polymorphisms, differences in spinal ABMC in late puberty were 2-fold greater in girls who were homozygous for the C and G alleles, respectively (P = 0.001). For rs7757956, the difference in total body less head ABMC in late puberty was 50% less in individuals homozygous or heterozygous for the A allele (P = 0.006). Conclusions: Gains in ABMC in late pubertal girls are strongly associated with ESR1 polymorphisms, suggesting that estrogen contributes to this process via an estrogen receptor α-dependent pathway.

Effect of an estrogen receptor-α intron 4 polymorphism on fat mass in 11-year-old children

Context: in the ESR1 gene encoding estrogen receptor (ER)-α may be associated with fat mass in adults. Objectives: The objective of the study was to establish whether ESR1 polymorphisms influence fat mass in childhood. Design: This was a cross-sectional analysis after genotyping of rs9340799, rs2234693, and rs7757956 ESR1 polymorphisms. Setting: The Avon Longitudinal Study of Parents and Children (ALSPAC) was a population-based prospective study. Participants: Participants included 3097 11-yr-old children with results for ESR1 genotyping, puberty measures, and dual-energy x-ray absorptiometry results. Outcomes: Relationships between ESR1 polymorphisms and indices of body composition were measured. Results: The rs7757956 polymorphism was associated with fat mass (P = 0.002). Total body fat mass (adjusted for height) was reduced by 6% in children with TA/AA genotypes, and risk of being overweight (≥85th centile of fat mass) was decreased by 20%. This genetic effect appeared to interact with puberty in girls (P = 0.05 for interaction): in those with the TT genotype, total body fat mass (adjusted for height) was 18% higher in Tanner stages 3-5 vs. stages 1-2; the equivalent difference was 7% in those with TA/AA genotypes. Furthermore, the risk of being overweight was 36% lower in girls with TA/AA genotypes in Tanner stages 3-5, but no reduction was seen in those in stages 1-2. Neither rs9340799 nor rs2234693 polymorphisms were associated with body composition measures. Conclusions: Fat mass in 11-yr-old children was related to the rs7757956 ESR1 polymorphism. This association was strongest in girls in more advanced puberty, in whom the risk of being overweight was reduced by 36% in those with the TA/AA genotype.

Influence of LRP5 polymorphisms on normal variation in BMD

Genetic studies based on cohorts with rare and extreme bone phenotypes have shown that the LRP5 gene is an important genetic modulator of BMD. Using family-based and case-control approaches, this study examines the role of the LRP5 gene in determining normal population variation of BMD and describes significant association and suggestive linkage between LRP5 gene polymorphisms and BMD in >900 individuals with a broad range of BMD. Introduction: Osteoporosis is a common, highly heritable condition determined by complex interactions of genetic and environmental etiologies. Genetic factors alone can account for 50-80% of the interindividual variation in BMD. Mutations in the LRP5 gene on chromosome 11q12-13 have been associated with rare syndromes characterized by extremely low or high BMD, but little is known about the contribution of this gene to the development of osteoporosis and determination of BMD in a normal population. Materials and Methods: To examine the entire spectrum of low to high BMD, 152 osteoporotic probands, their families (597 individuals), and 160 women with elevated BMD (T score > 2.5) were recruited. BMD at the lumbar spine, femoral neck, and hip were measured in each subject using DXA. Results: PAGE sequencing of the LRP5 gene revealed 10 single nucleotide polymorphisms (SNPs), 8 of which had allele frequencies of >5%, in exons 8, 9, 10, 15, and 18 and in introns 6, 7, and 21. Within families, a strong association was observed between an SNP at nucleotide C171346A in intron 21 and total hip BMD (p < 1 × 10-5 in men only, p = 0.0019 in both men and women). This association was also observed in comparisons of osteoporotic probands and unrelated elevated BMD in women (p = 0.03), along with associations with markers in exons 8 (C135242T, p = 0.007) and 9 (C141759T, p = 0.02). Haplotypes composed of two to three of the SNPs G121513A, C135242T, G138351A, and C141759T were strongly associated with BMD when comparing osteoporotic probands and high BMD cases (p < 0.003). An SNP at nucleotide C165215T in exon 18 was linked to BMD at the lumbar spine, femoral neck, and total hip (parametric LOD scores = 2.8, 2.5, and 2.2 and nonparametric LOD scores = 0.3, 1.1, and 2.2, respectively) but was not genetically associated with BMD variation. Conclusion: These results show that common LRP5 polymorphisms contribute to the determination of BMD in the general population.

Development of molecular tools for the improvement of transgene expression in sugar cane

Biotechnology has the potential to improve sugar cane, one of the world's major crops for food and fuel. This research describes the detailed characterisation of introns and their potential for enhancing transgene expression in sugar cane via intron-mediated enhancement (IME). IME is a phenomenon whereby an intron enhances gene expression from a promoter. Current knowledge on the mechanism of IME or its potential for enhancing gene expression in sugar cane is limited. A better understanding of the factors responsible for IME will help develop new molecular tools that facilitate high levels of constitutive and tissue-specific gene expression in this crop.

DNA structural variability as a factor in gene expression and evolution

Redundant DNA can buffer sequence dependent structural deviations from an ideal double helix. Buffering serves a mechanistic function by reducing extraneous conformational effects which could interfere with readout or which would impose energetic constraints on evolution. It also serves an evolutionary function by allowing for gradual variations in conformation-dependent regulation of gene expression. Such gradualism is critical for the rate of evolution. The buffer structure concept provides a new interpretation for repetitive DNA and for exons and introns.

Development of type I genetic markers from expressed sequence tags in highly polymorphic species

Expressed sequence tag (EST) databases provide a primary source of nuclear DNA sequences for genetic marker development in non-model organisms. To date, the process has been relatively inefficient for several reasons: - 1) priming site polymorphism in the template leads to inferior or erratic amplification; - 2) introns in the target amplicon are too large and/or numerous to allow effective amplification under standard screening conditions, and; - 3) at least occasionally, a PCR primer straddles an exon–intron junction and is unable to bind to genomic DNA template. The first is only a minor issue for species or strains with low heterozygosity but becomes a significant problem for species with high genomic variation, such as marine organisms with extremely large effective population sizes. Problems arising from unanticipated introns are unavoidable but are most pronounced in intron-rich species, such as vertebrates and lophotrochozoans. We present an approach to marker development in the Pacific oyster Crassostrea gigas, a highly polymorphic and intron-rich species, which minimizes these problems, and should be applicable to other non-model species for which EST databases are available. Placement of PCR primers in the 3′ end of coding sequence and 3′ UTR improved PCR success rate from 51% to 97%. Almost all (37 of 39) markers developed for the Pacific oyster were polymorphic in a small test panel of wild and domesticated oysters.

U12-type Spliceosome: Localization and Effects of Splicing Efficiency on Gene Expression

The removal of non-coding sequences, introns, is an essential part of messenger RNA processing. In most metazoan organisms, the U12-type spliceosome processes a subset of introns containing highly conserved recognition sequences. U12-type introns constitute less than 0,5% of all introns and reside preferentially in genes related to information processing functions, as opposed to genes encoding for metabolic enzymes. It has previously been shown that the excision of U12-type introns is inefficient compared to that of U2-type introns, supporting the model that these introns could provide a rate-limiting control for gene expression. The low efficiency of U12-type splicing is believed to have important consequences to gene expression by limiting the production of mature mRNAs from genes containing U12-type introns. The inefficiency of U12-type splicing has been attributed to the low abundance of the components of the U12-type spliceosome in cells, but this hypothesis has not been proven. The aim of the first part of this work was to study the effect of the abundance of the spliceosomal snRNA components on splicing. Cells with a low abundance of the U12-type spliceosome were found to inefficiently process U12-type introns encoded by a transfected construct, but the expression levels of endogenous genes were not found to be affected by the abundance of the U12-type spliceosome. However, significant levels of endogenous unspliced U12-type intron-containing pre-mRNAs were detected in cells. Together these results support the idea that U12-type splicing may limit gene expression in some situations. The inefficiency of U12-type splicing has also promoted the idea that the U12-type spliceosome may control gene expression, limiting the mRNA levels of some U12-type intron-containing genes. While the identities of the primary target genes that contain U12-type introns are relatively well known, little has previously been known about the downstream genes and pathways potentially affected by the efficiency of U12-type intron processing. Here, the effects of U12-type splicing efficiency on a whole organism were studied in a Drosophila line with a mutation in an essential U12-type spliceosome component. Genes containing U12-type introns showed variable gene-specific responses to the splicing defect, which points to variation in the susceptibility of different genes to changes in splicing efficiency. Surprisingly, microarray screening revealed that metabolic genes were enriched among downstream effects, and that the phenotype could largely be attributed to one U12-type intron-containing mitochondrial gene. Gene expression control by the U12-type spliceosome could thus have widespread effects on metabolic functions in the organism. The subcellular localization of the U12-type spliceosome components was studied as a response to a recent dispute on the localization of the U12-type spliceosome. All components studied were found to be nuclear indicating that the processing of U12-type introns occurs within the nucleus, thus clarifying a question central to the field. The results suggest that the U12-type spliceosome can limit the expression of genes that contain U12-type introns in a gene-specific manner. Through its limiting role in pre-mRNA processing, the U12-type splicing activity can affect specific genetic pathways, which in the case of Drosophila are involved in metabolic functions.

Expression of Human Growth Hormone in Silkworm Larvae through RecombinantBombyx moriNuclear Polyhedrosis Virus

We have generated a recombinantBombyx morinuclear polyhedrosis virus, vBmhGH, harboring the full-length human growth hormone gene (2.4-kb genomic DNA, with four introns and the signal peptide sequences) under the control of the polyhedrin promoter. BmN cells in culture infected with the recombinant virus showed the presence of RNA corresponding to the authentic growth hormone mRNA as well as its incompletly processed precusor. Electrophoretic analysis and immunoprecipitation of proteins of recombinant virus-infected BmN cells revealed the presence of the growth hormone protein. Infection of silkworm larvae with vBmhGH led to the synthesis and efficient secretion of the protein into hemolymph. The recombinant human growth hormone was biologically active in a radioreceptor competition binding assay. The secreted protein was isolated and purified to homogeneity by a single step immunoaffinity chromatography, to a specific activity of 2.4 × 104U/mg. The recombinant hGH retained the immunological and biolological properties of the native peptide. We conclude that BmNPV vectors can be used successfully for expressing chromosomal genes harboring multiple introns.

Structural and functional characteristics of homing endonucleases

Mobile genetic elements constitute a remarkably diverse group of nonessential selfish genes that provide no apparent function to the host. These selfish genes have been implicated in host extinction, speciation and architecture of genetic systems. Homing endonucleases, encoded by the open reading frames embedded in introns or inteins of mobile genetic elements, possess double-stranded DNA-specific endonuclease activity. They inflict sequence-specific double-strand breaks at or near the homing site in intron- or intein-less allele. Subsequently, through nonreciprocal exchange the insertion sequence (intron or intein) is transferred from an intein- or intron-containing allele to an intein- or intron-less allele. The components of host double-strand break repair pathway are thought to finish the "homing" process. Several lines of evidence suggest that homing endonucleases are capable of promoting transposition into ectopic sites within or across genomes for their survival as well as dispersal in natural populations. The occurrence of inteins at high frequencies serves as instructive models for understanding the mechanistic aspects of the process of homing and its evolution. This review focuses on genetic, biochemical, structural, and phylogenetic aspects of homing endonucleases, and their comparison with restriction endonucleases.

The RecA intein of Mycobacterium tuberculosis promotes cleavage of ectopic DNA sites. Implications for the dispersal of inteins in natural populations

The RecA intein of Mycobacterium tuberculosis, a novel double-stranded DNA endonuclease, requires both Mn(2+) and ATP for efficient cleavage of the inteinless recA allele. In this study, we show that Mg(2+) alone was sufficient to stimulate PI-MtuI to cleave double-stranded DNA at ectopic sites. In the absence of Mg(2+), PI-MtuI formed complexes with topologically different forms of DNA containing ectopic recognition sequences with equal affinity but failed to cleave DNA. We observed that PI-MtuI was able to inflict double-strand breaks robustly within the ectopic recognition sequence to generate either a blunt end or 1-2-nucleotide 3'-hydroxyl overhangs. Mutational analyses of the presumptive metal ion-binding ligands (Asp(122), Asp(222), and Glu(220)) together with immunoprecipitation assays provided compelling evidence to link both the Mg(2+)- and Mn(2+) and ATP-dependent endonuclease activities to PI-MtuI. The kinetic mechanism of PI-MtuI promoted cleavage of ectopic DNA sites proceeded through a sequential mechanism with transient accumulation of nicked circular duplex DNA as an intermediate. Together, these data suggest that PI-MtuI, like group II introns, might mediate ectopic DNA transposition and hence its lateral transfer in natural populations.

Ultra high throughput sequencing excludes MDH1 as candidate gene for RP28-linked retinitis pigmentosa

Purpose: Mutations in IDH3B, an enzyme participating in the Krebs cycle, have recently been found to cause autosomal recessive retinitis pigmentosa (arRP). The MDH1 gene maps within the RP28 arRP linkage interval and encodes cytoplasmic malate dehydrogenase, an enzyme functionally related to IDH3B. As a proof of concept for candidate gene screening to be routinely performed by ultra high throughput sequencing (UHTs), we analyzed MDH1 in a patient from each of the two families described so far to show linkage between arRP and RP28. Methods: With genomic long-range PCR, we amplified all introns and exons of the MDH1 gene (23.4 kb). PCR products were then sequenced by short-read UHTs with no further processing. Computer-based mapping of the reads and mutation detection were performed by three independent software packages. Results: Despite the intrinsic complexity of human genome sequences, reads were easily mapped and analyzed, and all algorithms used provided the same results. The two patients were homozygous for all DNA variants identified in the region, which confirms previous linkage and homozygosity mapping results, but had different haplotypes, indicating genetic or allelic heterogeneity. None of the DNA changes detected could be associated with the disease. Conclusions: The MDH1 gene is not the cause of RP28-linked arRP. Our experimental strategy shows that long-range genomic PCR followed by UHTs provides an excellent system to perform a thorough screening of candidate genes for hereditary retinal degeneration.

Cloning by genomic PCR and production of peanut agglutinin in Escherichia coli

Using the polymerase chain reaction, the coding sequence for peanut agglutinin (PNA) was cloned and expressed in Escherichia coli. Amplified PNA is identical to previously reported cDNA, suggesting the absence of any introns in PNA gene. Recombinant (re-) PNA forms inclusion bodies in E. coli. Production of PNA was confirmed by probing Western blots with polyclonal anti-PNA immunoglobulin G. Inclusion bodies were solubilized with 6 M guanidine-HCl and renatured by rapid dilution in the presence of metal ions. The renatured lectin was then purified by affinity chromatography. The re-lectin shows carbohydrate-binding properties similar to the natural PNA. This expression system provides a model for future mutagenesis studies of the carbohydrate-binding site and thus facilitates ongoing efforts to explore the molecular basis for the specificity of lectin-carbohydrate interaction.

Ultra high throughput sequencing excludes MDH1 as candidate gene for RP28-linked retinitis pigmentosa

Purpose: Mutations in IDH3B, an enzyme participating in the Krebs cycle, have recently been found to cause autosomal recessive retinitis pigmentosa (arRP). The MDH1 gene maps within the RP28 arRP linkage interval and encodes cytoplasmic malate dehydrogenase, an enzyme functionally related to IDH3B. As a proof of concept for candidate gene screening to be routinely performed by ultra high throughput sequencing (UHTs), we analyzed MDH1 in a patient from each of the two families described so far to show linkage between arRP and RP28. Methods: With genomic long-range PCR, we amplified all introns and exons of the MDH1 gene (23.4 kb). PCR products were then sequenced by short-read UHTs with no further processing. Computer-based mapping of the reads and mutation detection were performed by three independent software packages. Results: Despite the intrinsic complexity of human genome sequences, reads were easily mapped and analyzed, and all algorithms used provided the same results. The two patients were homozygous for all DNA variants identified in the region, which confirms previous linkage and homozygosity mapping results, but had different haplotypes, indicating genetic or allelic heterogeneity. None of the DNA changes detected could be associated with the disease.

Hiekkatympösen (Hebeloma cylindrosporum) L-aminohappo-oksidaasi -geenin eristäminen

Pohjoisella havumetsävyöhykkeellä typpi on usein kasvien kasvua rajoittava tekijä. Metsämaan typpivarannot koostuvat pääasiassa orgaaniseen ainekseen sitoutuneista typpiyhdisteistä, erityisesti aminohapoista. Ektomykorritsasienet osallistuvat metsämaassa tapahtuvaan typenkiertoon hajottamalla orgaanisia typpiyhdisteitä ja kuljettamalla niitä kasvien käytettäväksi. Sienisolun sisällä tapahtuvasta aminohappojen mineralisaatiosta tiedetään toistaiseksi melko vähän. Aminohappo-oksidaasit katalysoivat aminohappojen mineralisaatiota. Eräissä ektomykorritsaa muodostavien kantasienten suvuissa on osoitettu L-aminohappo-oksidaaseja (LAO). Toistaiseksi LAO-geeniä ei tunneta kantasienistä. Työssä kuvattiin ensimmäistä kertaa LAO-geeni kantasienistä. Hiekkatympösen LAO1- geenin cDNA:n 5´ ja 3´ päiden emäsjärjestykset määritettiin RACE-PCR -menetelmällä, josta saatujen sekvenssien perusteella suunniteltiin alukkeet koko geenin cDNA:n ja genomisen DNA:n monistamiseksi. Genomisen DNA ja cDNA -sekvenssien perusteella määritettiin hiekkatympösen LAO1-geenin rakenne. Hiekkatympösen LAO1-geeni koostuu viidestä eksonista ja neljästä intronista. Hiekkatympösen LAO1-geenin yläpuoliselta alueelta löydettiin typpimetabolian säätelyyn osallistuvan proteiinin sitoutumiskohta. LAO1-geeniä edeltävä geenin osittainen genominen DNA-sekvenssi määritettiin. Kangaslohisienen genomissa LAO1-geeniä edeltävä geeni oli ennustettu pyruvaattidekarboksylaasiksi. Lisäksi työssä määritettiin hiekkatympösen toisen LAOhomologin cDNA:n osittainen emäsjärjestys. Työssä tunnistettiin myös toisen kantasienen, kangaslohisienen, LAO-geeni. LAO-geeniksi tunnistettu kangaslohisienen geenimalli oli aiemmin ennustettu NCBI:n tietokannassa toiminnaltaan tuntemattomaksi proteiiniksi. Proteiinien sukupuun perusteella hiekkatympösen ja kangaslohisienen LAO:n kantamuoto on kahdentunut. Työstä saatu tutkimustulos tuo täysin uutta tietoa molekyylibiologian tasolla ektomykorritsasienten aminohappojen katabolisista reaktioista. Aminohappojen mineralisaation seurauksen muodostuneet ammoniumionit saattavat olla merkittävä typen lähde myös maan muille mikrobeille ja kasveille. On mahdollista, että ektomykorritsasienten LAO-entsyymi on yksi merkittävä tekijä metsämaan typenkierrossa.

The function and regulation of the U11-48K protein in U12-dependent splicing

The removal of noncoding sequences, or introns, from the eukaryotic messenger RNA precursors is catalyzed by a ribonucleoprotein complex known as the spliceosome. In most eukaryotes, two distinct classes of introns exist, each removed by a specific type of spliceosome. The major, U2-type introns account for over 99 % of all introns, and are almost ubiquitous. The minor, U12-type introns are found in most but not all eukaryotes, and reside in conserved locations in a specific set of genes. Due to their slow excision rates, the U12-type introns are expected to be involved in the regulation of the genes containing them by inhibiting the maturation of the messenger RNAs. However, little information is currently available on how the activity of the U12-dependent spliceosome itself is regulated. The levels of many known splicing factors are regulated through unproductive alternative splicing events, which lead to inclusion of premature STOP codons, targeting the transcripts for destruction by the nonsense-mediated decay pathway. These alternative splice sites are typically found in highly conserved sequence elements, which also contain binding sites for factors regulating the activation of the splice sites. Often, the activation is achieved by binding of products of the gene in question, resulting in negative feedback loops. In this study, I show that U11-48K, a protein factor specific to the minor spliceosome, specifically recognizes the U12-type 5' splice site sequence, and is essential for proper function of the minor spliceosome. Furthermore, the expression of U11-48K is regulated through a feedback mechanism, which functions through conserved sequence elements that activate alternative splicing and nonsense-mediated decay. This mechanism is conserved from plants to animals, highlighting both the importance and early origin of this mechanism in regulating splicing factors. I also show that the feedback regulation of U11-48K is counteracted by a component of the major spliceosome, the U1 small nuclear ribonucleoprotein particle, as well as members of the hnRNP F/H protein family. These results thus suggest that the feedback mechanism is finely tuned by multiple factors to achieve precise control of the activity of the U12-dependent spliceosome.