ABP1 Gene Research Articles

Fluorescence Marker-Assisted Isolation of Cas9-Free and CRISPR-Edited Arabidopsis Plants.

CRISPR/Cas9 gene editing technology has successfully introduced modifications at target DNA sequences in many plant species including Arabidopsis. After the target gene is edited, the CRISPR/Cas9 construct needs to be removed to ensure genetic stability and to gain any regulatory approval for commercial applications. However, removal of the transgenes by genetic segregation, backcross, and genotyping is very laborious and time-consuming. The methods we report here allow fast and effective isolation of transgene-free T2 Arabidopsis plants with the desired modifications at the target genes. We express a fluorescence protein mCherry under the control of a seed-specific promoter At2S3 and placed the cassette into the CRISPR/Cas9 vector. Therefore, we can use mCherry as a proxy for the presence of Cas9, and we are able to visually isolate the Cas9-free Arabidopsis plants with heritable mutations at the T2 generation. We targeted two sites in the ABP1 gene to demonstrate the effectiveness of our approach.

Disrupting the cortical actin cytoskeleton points to two distinct mechanisms of yeast [PSI+] prion formation.

Mammalian and fungal prions arise de novo; however, the mechanism is poorly understood in molecular terms. One strong possibility is that oxidative damage to the non-prion form of a protein may be an important trigger influencing the formation of its heritable prion conformation. We have examined the oxidative stress-induced formation of the yeast [PSI+] prion, which is the altered conformation of the Sup35 translation termination factor. We used tandem affinity purification (TAP) and mass spectrometry to identify the proteins which associate with Sup35 in a tsa1 tsa2 antioxidant mutant to address the mechanism by which Sup35 forms the [PSI+] prion during oxidative stress conditions. This analysis identified several components of the cortical actin cytoskeleton including the Abp1 actin nucleation promoting factor, and we show that deletion of the ABP1 gene abrogates oxidant-induced [PSI+] prion formation. The frequency of spontaneous [PSI+] prion formation can be increased by overexpression of Sup35 since the excess Sup35 increases the probability of forming prion seeds. In contrast to oxidant-induced [PSI+] prion formation, overexpression-induced [PSI+] prion formation was only modestly affected in an abp1 mutant. Furthermore, treating yeast cells with latrunculin A to disrupt the formation of actin cables and patches abrogated oxidant-induced, but not overexpression-induced [PSI+] prion formation, suggesting a mechanistic difference in prion formation. [PIN+], the prion form of Rnq1, localizes to the IPOD (insoluble protein deposit) and is thought to influence the aggregation of other proteins. We show Sup35 becomes oxidized and aggregates during oxidative stress conditions, but does not co-localize with Rnq1 in an abp1 mutant which may account for the reduced frequency of [PSI+] prion formation.

Genetic variation within the histamine pathway among patients with asthma – a pilot study

Objective: Histamine is an important mediator in the pathophysiology of asthma. We have previously reported that HRH1 is differentially expressed among those with asthma compared to those without asthma. Single histamine-related genes have also been associated with asthma. We aimed to evaluate known single nucleotide polymorphisms (SNPs) in genes along the histamine biotransformation and response pathway, and determine their association with asthma and HRH1 mRNA expression. Methods: We enrolled children and adults (n = 93) with/without asthma who met inclusion/exclusion criteria. Genotyping was performed for nine known SNPs in the HDC, HRH1, HRH4, HNMT and ABP1 genes. HRH1 mRNA expression was determined on RNA from buccal tissue. General linear model, Fisher’s exact test and Chi-square test were used to determine differences in allele, genotype and haplotype frequency between subjects with and without asthma and differential HRH1 mRNA expression relative to genotype. Statistical significance was determined by p < 0.05. Results: No difference was observed in genotype/allele frequency for the nine SNPs between subjects with and without asthma. The HNMT-1639C/-464C/314C/3′UTRA haplotype was more frequently observed in those without asthma than those with asthma (p = 0.03). We also observed genetic differences relative to race and gender. HNMT 314 genotype CT was more frequent in males with asthma compared to those without asthma (p = 0.04). Conclusions: Histamine pathway haplotype was associated with a diagnosis of asthma in our cohort but allele and genotype were not. Subgroup evaluations may also be important. Further studies are needed to determine the potential biological/clinical significance of our findings.

Identification of rare DNA sequence variants in high-risk autism families and their prevalence in a large case/control population

BackgroundGenetics clearly plays a major role in the etiology of autism spectrum disorders (ASDs), but studies to date are only beginning to characterize the causal genetic variants responsible. Until recently, studies using multiple extended multi-generation families to identify ASD risk genes had not been undertaken.MethodsWe identified haplotypes shared among individuals with ASDs in large multiplex families, followed by targeted DNA capture and sequencing to identify potential causal variants. We also assayed the prevalence of the identified variants in a large ASD case/control population.ResultsWe identified 584 non-conservative missense, nonsense, frameshift and splice site variants that might predispose to autism in our high-risk families. Eleven of these variants were observed to have odds ratios greater than 1.5 in a set of 1,541 unrelated children with autism and 5,785 controls. Three variants, in the RAB11FIP5, ABP1, and JMJD7-PLA2G4B genes, each were observed in a single case and not in any controls. These variants also were not seen in public sequence databases, suggesting that they may be rare causal ASD variants. Twenty-eight additional rare variants were observed only in high-risk ASD families. Collectively, these 39 variants identify 36 genes as ASD risk genes. Segregation of sequence variants and of copy number variants previously detected in these families reveals a complex pattern, with only a RAB11FIP5 variant segregating to all affected individuals in one two-generation pedigree. Some affected individuals were found to have multiple potential risk alleles, including sequence variants and copy number variants (CNVs), suggesting that the high incidence of autism in these families could be best explained by variants at multiple loci.ConclusionsOur study is the first to use haplotype sharing to identify familial ASD risk loci. In total, we identified 39 variants in 36 genes that may confer a genetic risk of developing autism. The observation of 11 of these variants in unrelated ASD cases further supports their role as ASD risk variants.

Auxin binding proteins ABP1 and ABP4 are involved in the light- and auxin-induced down-regulation of phytochrome gene PHYB in maize (Zea mays L.) mesocotyl

Previous research in maize suggested a possible involvement of auxin-binding proteins (ABPs) in light signaling during maize seedling development. To obtain more information about the interaction of auxin and light signaling, we investigated the gene expression of phytochrome B (PHYB) and phytochrome A (PHYA) in loss-of-function mutants in ABP1 and ABP4 genes in maize (Zea mays L.). We studied how expression of the PHYB and PHYA genes in mesocotyl is regulated by white light (WL), and whether exogenous auxin NAA influences the expression of the phytochrome genes. We found that knockout of ABP1 or ABP4 results in essentially reduced expression of PHYB gene in dark-grown mesocotyl. WL reduced PHYB expression in WT but not in the ABPs knockout seedlings. The data indicate that ABP1 and ABP4 are positively involved in PHYB expression in etiolated mesocotyl. Our results also indicate that in etiolated mesocotyl, ABP1 and ABP4 mediate the inhibitory effect of exogenous auxin on level of PHYB transcript. In contrast, in our experimental conditions, WL does not reduce expression of PHYA. Our results further suggest that ABP1 and ABP4 are not likely involved in the expression of PHYA gene and neither in auxin-induced suppression of PHYA transcript accumulation. Our results support the existence of cross-talk between auxin and light signaling and indicate for the first time that ABP1, ABP4 and PHYB genes could share common signaling pathway(s).

Going in the Right Direction: Mating-Type Switching ofSchizosaccharomyces pombeIs Controlled by Judicious Expression of Two Differentswi2Transcripts

Schizosaccharomyces pombe, the fission yeast, cells alternate between P- and M-mating type, controlled by the alternate alleles of the mating-type locus (mat1). The mat1 switching occurs by replacing mat1 with a copy derived from a silenced "donor locus," mat2P or mat3M. The mechanism of donor choice ensuring that switching occurs primarily and productively to the opposite type, called directionality, is largely unknown. Here we identified the mat1-Mc gene, a mammalian sex-determination gene (SRY) homolog, as the primary gene that dictates directionality in M cells. A previously unrecognized, shorter swi2 mRNA, a truncated form of the swi2, was identified, and its expression requires the mat1-Mc function. We also found that the abp1 gene (human CENPB homolog) controls directionality through swi2 regulation. In addition, we implicated a cis-acting DNA sequence in mat2 utilization. Overall, we showed that switching directionality is controlled by judicious expression of two swi2 transcripts through a cell-type-regulated dual promoter. In this respect, this regulation mechanism resembles that of the Drosophila sex-determination Slx gene.

Polymorphisms of two histamine-metabolizing enzymes genes and childhood allergic asthma: a case control study

BackgroundHistamine-metabolizing enzymes (N-methyltransferase and amiloride binding protein 1) are responsible for histamine degradation, a biogenic amine involved in allergic inflammation. Genetic variants of HNMT and ABP1 genes were found to be associated with altered enzyme activity. We hypothesized that alleles leading to decreased enzyme activity and, therefore, decreased inactivation of histamine may be responsible for altered susceptibility to asthma.MethodsThe aim of this study was to analyze polymorphisms within the HNMT and ABP1 genes in the group of 149 asthmatic children and in the group of 156 healthy children. The genetic analysis involved four polymorphisms of the HNMT gene: rs2071048 (-1637T/C), rs11569723 (-411C/T), rs1801105 (Thr105Ile = 314C/T) and rs1050891 (1097A/T) and rs1049793 (His645Asp) polymorphism for ABP1 gene. Genotyping was performed with use of PCR-RFLP. Statistical analysis was performed using Statistica software; linkage disequilibrium analysis was done with use of Haploview software.ResultsWe found an association of TT genotype and T allele of Thr105Ile polymorphism of HNMT gene with asthma. For other polymorphisms for HNMT and ABP1 genes, we have not observed relationship with asthma although the statistical power for some SNPs might not have been sufficient to detect an association. In linkage disequilibrium analysis, moderate linkage was found between -1637C/T and -411C/T polymorphisms of HNMT gene. However, no significant differences in haplotype frequencies were found between the group of the patients and the control group.ConclusionsOur results indicate modifying influence of histamine N-methyltransferase functional polymorphism on the risk of asthma. The other HNMT polymorphisms and ABP1 functional polymorphism seem unlikely to affect the risk of asthma.

Effect of auxin on mesocotyl elongation of dark-grown maize under different seeding depths

In order to elucidate the physiological mechanism of maize mesocotyl elongation induced by auxin under different seeding depths, seeds of five maize inbred lines, including 3681-4 line tolerant to deep seeding, were treated with IAA and triiodobenzoic acid (TIBA) under seeding depths of 20 or 2 cm. Under deep seeding conditions, maize mesocotyls grew by 1.5–2.0 times faster than under shallow seeding conditions. IAA (10−6 to 10−4 M) applied to roots stimulated mesocotyl elongation only of 3681-4 line and only under deep seeding conditions. TIBA (10−5 and 10−4 M) applied to roots inhibited mesocotyl elongation in all lines, but only 3681-4 was sensitive to 10−6 M TIBA. IAA promoted only cell elongation, and TIBA inhibited both cell elongation and cell division. After IAA and TIBA treatments, endogenous IAA content changed in parallel with the mesocotyl growth rate under different seeding depths. Furthermore, ABP1 gene expression changed in parallel with the mesocotyl growth rate under deep seeding conditions. Therefore, deep seeding tolerance of 3681-4 line was achieved due to auxin-regulated rapid mesocotyl elongation.

P236 - MEFV mutations modify the disease severity of inflammatory bowel disease

binding protein 1, ABP1; EC 1.4.3.6). ABP1 enzyme activity in bowel mucosa of patients with IBD is 50% lower than in healthy individuals. The reason for this decrease remains unknown, but it may be due in part to polymorphisms in the ABP1 gene. The present study investigated whether a common single nucleotide polymorphism (SNP, refSNP ID: rs1049793), which is located in exon 3 C2029G) and cause amino acid substitutions (His645Asp) in the ABP1 enzyme, is related to CD. Aims: To analyse the association between the presence of a non-synonymous single nucleotide polymorphism at diamine oxidase gene and the risk of developing Crohn’s disease (CD) and to analyse its influence on the clinical course of these patients. Methods: In this prospective, case-control study, 210 unrelated Caucasian consecutive CD patients were recruited at the Inflammatory Bowel Disease Unit of a single tertiary centre (Hospital Clinico San Carlos) in Madrid, Spain. All patients were phenotyped and followed up for a median time of 8.7 years (range 4.1 14.5 years). A total of 261 healthy volunteers from the same geographic area were also recruited and matched with patients. Both cases and controls were analysed for the presence of His645Asp amino acid substitutions in the diamine oxidase enzyme, using amplification-restriction procedures. The protocol was approved by the Ethics Committee of the Hospital and all patients and controls gave informed consent before inclusion in the study. Results: No significant differences were found in the distribution of ABP1 alleles between CD patients and healthy volunteers [for variant alleles = OR 1.15 (95%CI 0.86 1.55)]. The distribution of ABP1 genotypes did not differ when patients were subdivided according to gender, mean age at diagnosis, mean duration of the disease, family history of IBD, smoking habit, previous appendectomy or tonsillectomy, Montreal classification, perianal CD, extraintestinal manifestations of CD (cutaneous, articular, ocular and hepatic) and severity, i.e. need for immunosuppressive therapies, biological treatments and/or surgery. Conclusion: Our results suggest that the His645Asp polymorphism of the histamine metabolising enzyme ABP1 may not be related to the risk of developing CD. Moreover, this gene does not seem to play a role in disease activity.

Genetic variability of human diamine oxidase: occurrence of three nonsynonymous polymorphisms and study of their effect on serum enzyme activity

To analyze the occurrence and the functional effects of nonsynonymous single nucleotide polymorphisms in the human diamine oxidase (ABP1) gene. Genomic DNA from 134 healthy Caucasian individuals was analyzed for three nonsynonymous single nucleotide polymorphisms in the ABP1 gene. Serum diamine oxidase activity was studied in 37 individuals with known ABP1 genotype. Variant ABP1 alleles leading to the amino-acid substitutions Thr16Met, Ser332Phe and His645Asp were identified with frequencies of 25.4, 6.3 and 30.6%, respectively. Over 70% of the population (95% confidence interval, 62.4-77.9%) carry at least one amino-acid substitution. Each amino-acid substitution was at Hardy-Weinberg's equilibrium, but linkage disequilibrium between variant alleles was observed. The percentage of individuals carrying simultaneously the three amino-acid substitutions in heterozygosity or homozygosity (9%, 95% confidence interval, 4.2-13.8%) was over three times that expected from a random association (P<0.05). Individuals carrying the 645Asp amino acid displayed lower serum diamine oxidase activity as compared with noncarriers (P<0.001) with a significant gene-dose effect (P<0.05). This was due to an increase in the Michaelis-Menten constant. Individuals heterozygous for 645Asp show Vmax/Km values of 66% and homozygous 51% as compared with noncarriers. The effect of the 16Met variant allele was lower and that of the rarest allele 332Phe was negligible. Nonsynonymous ABP1 gene polymorphisms are common in humans; they cause relevant functional effects and can be considered as major determinants of variability for human diamine oxidase activity.

Molecular characterization and spatial expression of the sunflower ABP1 gene.

We have used RT-PCR and low-stringency cDNA library screening to isolate the coding sequence of the sunflower auxin-binding protein (ABP1). All the clones analysed contained the same nucleotide sequence, suggesting that ABP1 is encoded by a single-copy gene in sunflower. The deduced amino acid sequence shows a high degree of similarity with ABP1 proteins from other plant species. Most remarkably, the sunflower protein lacks two cysteine residues present in all other plant ABPs known to date and shown to be involved in a disulfide bridge in the maize protein. Genomic Southern hybridization data support the existence of a single copy of the ABP1 gene in the sunflower genome. Northern hybridization corroborated earlier observations indicating that the steady-state level of ABP1 transcript is higher in actively dividing and growing organs than in the rest of the plant: it is more abundant in the shoot apex, floral buds and immature embryos than in mature leaves, stem, roots and ray flowers. To characterize the tissular ABP1 transcript distribution in sunflower, various organ sections were analysed upon in situ hybridization. Localized accumulation of the ABP1 transcript suggests that its spatial expression is highly regulated at the tissue level. In addition, the transcript preferentially accumulates in tissues having a high rate of cellular division, such as shoot and root apical meristems, leaf primordia and pro-vascular tissues. The ABP1 expression pattern was also studied at a temporal scale during lateral root formation. Real time PCR showed an elevation of the steady state level of the ABP1 transcript in root axes after 36 h of seed germination. In situ hybridization revealed that this global increase is the result of local accumulation of the ABP1 transcript in lateral root primordia, which are known to develop under auxin action. The possibility that a high ABP1 expression level correlates with a high cellular sensitivity to auxin is discussed.

Crystallization and preliminary X-ray analysis of the auxin receptor ABP1.

Auxin-binding protein (ABP1) is an important receptor for the plant hormone auxin that is involved with many growth and developmental responses in plants. The maize ABP1 gene has been expressed in insect cells, purified and crystallized. Type II crystals are monoclinic, with two glycosylated homodimers in the asymmetric unit, and diffract to 1.9 A using synchrotron radiation.

Molecular characterization of the Abp1 5'-flanking region in maize and the teosintes.

Auxin-binding protein 1 subsp. mays (ABP1) has been suggested as a receptor mediating auxin-induced cell expansion and differentiation. In maize (Zea mays), ABP1 is encoded by a single gene, Abp1. The TATA and CAAT promoter elements as well as the transcriptional start site were previously identified and all were found to be located within a transposable element (TE), Tourist-Zm11. In this study we report the cloning and characterization of the Abp1 5'-flanking region in maize and its wild relatives, the teosintes. We provide evidence for insertion polymorphism corresponding to Tourist-Zm11 and two novel TEs, Batuta and Pilgrim. Despite this polymorphic structure, the Abp1 core promoter in maize and the teosintes is conserved, is located downstream of the TE insertions in the 5'-flanking region, and is TATA-less. We discuss the potential evolutionary impact of these TEs on the regulation of Abp1 gene expression.

Cloning and expression of two genes encoding auxin-binding proteins from tobacco.

Two genes encoding the auxin-binding protein (ABP1) of tobacco (Nicotiana tabacum L.), both of which possess the characteristics of a luminal protein of the endoplasmic reticulum (ER), were isolated and sequenced. These genes were composed of at least five exons and four introns. The two coding exons showed 95% sequence homology and coded for two precursor proteins of 187 amino acid residues with molecular masses of 21,256 and 21,453 Da. The deduced amino acid sequences were 93% identical and both possessed an amino-terminal signal peptide, a hydrophilic mature protein region with two potential N-glycosylation sites and a carboxyl-terminal sorting signal, KDEL, for the ER. Restriction mapping of the cDNAs encoding tobacco ABP1, previously purified by amplification of tobacco cDNA libraries by polymerase chain reaction (PCR) using specific primers common to both genes, indicated that both genes were expressed, although one was expressed at a higher level than the other. Genomic Southern blot hybridization showed no other homologous genes except for these two in the tobacco genome. The apparent molecular mass of the mature form of tobacco ABP1 was revealed to be 25 kDa by SDS polyacrylamide gel electrophoresis using affinity-purified anti (tobacco ABP1) antibodies raised against a fusion protein with maltose-binding protein. Expression of the recombinant ABP1 gene in transgenic tobacco resulted in accumulation of the 25 kDa protein. A single point mutation of an amino acid residue at either of the two potential N-glycosylation sites resulted in a decrease in the apparent molecular mass and produced a 22 kDa protein. Mutations at both sites resulted in the formation of a 19.3 kDa protein, suggesting that tobacco ABP1 is glycosylated at two asparagine residues.

A centromere DNA-binding protein from fission yeast affects chromosome segregation and has homology to human CENP-B.

Genetic and biochemical strategies have been used to identify Schizosaccharomyces pombe proteins with roles in centromere function. One protein, identified by both approaches, shows significant homology to the human centromere DNA-binding protein, CENP-B, and is identical to Abp1p (autonomously replicating sequence-binding protein 1) (Murakami, Y., J.A. Huberman, and J. Hurwitz. 1996. Proc. Natl. Acad. Sci. USA. 93:502-507). Abp1p binds in vitro specifically to at least three sites in centromeric central core DNA of S. pombe chromosome II (cc2). Overexpression of abp1 affects mitotic chromosome stability in S. pombe. Although inactivation of the abp1 gene is not lethal, the abp1 null strain displays marked mitotic chromosome instability and a pronounced meiotic defect. The identification of a CENP-B-related centromere DNA-binding protein in S. pombe strongly supports the hypothesis that fission yeast centromeres are structurally and functionally related to the centromeres of higher eukaryotes.

Stable expression of maize auxin-binding protein in insect cell lines

To achieve continuous expression of the major maize auxin-binding protein (ABP1) in insect cells, the ABP1 gene coding region was placed under control of a baculovirus immediate-early gene promoter and transfected into Spodoptera frugiperda Sf9 cells. The ABP1 gene was detected in twelve cell lines, one of which was selected for detailed analysis. Immunolocalisation demonstrated that ABP1 was targeted to and retained in the endoplasmic reticulum (ER), in accordance with its signal peptide and car☐y-terminal KDEL ER-retention signal. We discuss the advantages of stable-transformation over transient expression systems for characterising proteins targeted to the secretory system of insect cells.

Mapping actin surfaces required for functional interactions in vivo.

An in vivo strategy to identify amino acids of actin required for functional interactions with actin-binding proteins was developed. This approach is based on the assumption that an actin mutation that specifically impairs the interaction with an actin-binding protein will cause a phenotype similar to a null mutation in the gene that encodes the actin-binding protein. 21 actin mutations were analyzed in budding yeast, and specific regions of actin subdomain 1 were implicated in the interaction with fimbrin, an actin filament-bundling protein. Mutations in this actin subdomain were shown to be, like a null allele of the yeast fimbrin gene (SAC6), lethal in combination with null mutations in the ABP1 and SLA2 genes, and viable in combination with a null mutation in the SLA1 gene. Biochemical experiments with act1-120 actin (E99A, E100A) verified a defect in the fimbrin-actin interaction. Genetic interactions between mutant alleles of the yeast actin gene and null alleles of the SAC6, ABP1, SLA1, and SLA2 genes also demonstrated that the effects of the 21 actin mutations are diverse and allowed four out of seven pseudo-wild-type actin alleles to be distinguished from the wild-type gene for the first time, providing evidence for functional redundancy between different surfaces of actin.