RESUMO
This study examined the amino acid sequence of the VIRESCENS gene (VIR), which regulates the production of anthocyanin in 12 cultivars of the date palm (Phoenix dactylifera L.), grown in Al-Madinah Al-Munawarah of the Kingdom of Saudi Arabia. The gene products were amplified via polymerase chain reactions, amplifying both exons and introns. The products were sequenced for the reconstruction of a phylogenetic tree, which used the associated amino acid sequences. The ripening stages of Khalal, Rutab, and Tamar varied among the cultivars. Regarding VIR genotype, the red date had the wild-type gene (VIR+), while the yellow date carried a dominant mutation (VIRIM), i.e., long terminal repeat retrotransposons (LTR-RTs). The DNA sequence of VIRIM revealed that the insertion length of the LTR-RTs ranged between 386 and 476 bp. The R2 and R3 motifs in both VIR+ and VIRIM were conserved. The C-terminus motifs S6A, S6B, and S6C were found in the VIR+ protein sequence. However, the amino acids at positions 123, 161, 166, and 168 differed between VIR+ and VIRIM, and were not included in the C-terminus motifs. Within the VIR+ allele, the lysine at position 187 in the C-terminus was located immediately after S6B, with a protein binding score of 0.3, which was unique to the dark, red-fruited cultivars Ajwah, Anbarah, and Safawi. In the lighter, red-fruited cultivars, the presence of glutamic acid at the same position suggested that the anthocyanin regulation of date palm might be outside the R2 and R3 domains in the N-terminus.
Assuntos
Phoeniceae , Phoeniceae/química , Sequência de Aminoácidos , Antocianinas/genética , Antocianinas/metabolismo , Filogenia , Reação em Cadeia da PolimeraseRESUMO
A novel series of bis- (Abdelhamid et al., 2017, Banerjee et al., 2018, Bharanidharan et al., 2022)thiadiazoles was synthesized from the reaction of precursor dimethyl 2,2'-(1,2-diphenylethane-1,2-diylidene)-bis(hydrazine-1-carbodithioate) and hydrazonyl chlorides in ethanol under ultrasonic irradiation. Spectral tools (IR. NMR, MS, elemental analyses, molecular dynamic simulation, DFT and LUMO and HOMO) were used to elucidate the structure of the isolated products. Molecular docking for the precursor, 3 and ligands 6a-i to two COVID-19 important proteins Mpro and RdRp was compared with two approved drugs, Remdesivir and Ivermectin. The binding affinity varied between the ligands and the drugs. The highest recorded binding affinity of 6c with Mpro was (-9.2 kcal/mol), followed by 6b and 6a, (-8.9 and -8.5 kcal/mol), respectively. The lowest recorded binding affinity was (-7.0 kcal/mol) for 6 g. In comparison, the approved drugs showed binding affinity (-7.4 and -7.7 kcal/mol), for Remdesivir and Ivermectin, respectively, which are within the range of the binding affinity of our ligands. The binding affinity of the approved drug Ivermectin against RdRp recoded the highest (-8.6 kcal/mol), followed by 6a, 6 h, and 6i are the same have (-8.2 kcal/mol). The lowest reading was found for compound 3 ligand (-6.3 kcal/mol). On the other side, the amino acids also differed between the compounds studied in this project for both the viral proteins. The ligand 6a forms three H-bonds with Thr 319(A), Sr 255(A) and Arg 457(A), whereas Ivermectin forms three H-bonds with His 41(A), Gly143(A) and Gln 18(A) for viral Mpro. The RdRp amino acids residues could be divided into four groups based on the amino acids that interact with hydrogen or hydrophobic interactions. The first group contained 6d, 6b, 6 g, and Remdesivir with 1-4 hydrogen bonds and hydrophobic interactions 1 to 10. Group 2 is 6a and 6f exhibited 1 and 3 hydrogen bonds and 15 and 14 hydrophobic interactions. Group 3 has 6e and Ivermectin shows 4 and 3 hydrogen bonds, respectively and 11 hydrophobic interactions for both compounds. The last group contains ligands 3, 6c, 6 h, and 6i gave 1-3 hydrogen bonds and 6c and 3 recorded the highest number of hydrophobic interactions, 14 for both 6c and 6 h. Pro Tox-II estimated compounds' activities as Hepatoxic, Carcinogenic and Mutagenic, revealing that 6f-h were inactive in all five similar to that found with Remdesivir and Ivermectin. The drug-likeness prediction was carried out by studying physicochemical properties, lipophilicity, size, polarity, insolubility, unsaturation, and flexibility. Generally, some properties of the ligands were comparable to that of the standards used in this study, Remdesivir and Ivermectin.
RESUMO
A novel series of 1-aryl-N-[4-phenyl-5-(arylazo)thiazol-2-yl)methanimines has been synthesized via the condensation of 2-amino-4-phenyl-5-arylazothiazole with various aromatic aldehydes. The synthesized imines were characterized by spectroscopic techniques, namely 1H and 13C-NMR, FTIR, MS, and Elemental Analysis. A molecular comparative docking study for 3a-f was calculated, with reference to two approved drugs, Molnupiravir and Remdesivir, using 7BQY (Mpro; PDB code 7BQY; resolution: 1.7 A°) under identical conditions. The binding scores against 7BQY were in the range of -7.7 to -8.7 kcal/mol for 3a-f. The high scores of the compounds indicated an enhanced binding affinity of the molecules to the receptor. This is due to the hydrophobic interactions and multi-hydrogen bonds between 3a-f ligands and the receptor's active amino acid residues. The main aim of using in silco molecular docking was to rank 3a-f with respect to the approved drugs, Molnupiravir and Remdesivir, using free energy methods as greener pastures. A further interesting comparison presented the laydown of the ligands before and after molecular docking. These results and other supporting statistical analyses suggested that ligands 3a-f deserve further investigation in the context of potential therapeutic agents for COVID-19. Free-cost, PASS, SwissADME, and Way2drug were used in this research paper to determine the possible biological activities and cytotoxicity of 3a-f.
Assuntos
Antivirais/química , Tratamento Farmacológico da COVID-19 , Iminas/química , Tiazóis/química , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/química , Alanina/análogos & derivados , Alanina/química , Antivirais/síntese química , Antivirais/farmacocinética , Antivirais/toxicidade , Sítios de Ligação , Simulação por Computador , Proteases 3C de Coronavírus/química , Citidina/análogos & derivados , Citidina/química , Hidroxilaminas/química , Iminas/síntese química , Iminas/farmacocinética , Iminas/toxicidade , Simulação de Acoplamento Molecular , SARS-CoV-2/efeitos dos fármacos , Tiazóis/síntese química , Tiazóis/farmacocinética , Tiazóis/toxicidadeRESUMO
This study reports the synthesis, characterization and importance of a novel diethyl 2-(2-(2-(3-methyl-2-oxoquinoxalin-1(2H)-yl)acetyl)hydrazono)malonate (MQOAHM). Two independent molecular structures of the disordered MQOAHM have been established by XRDsinglecrystal analysis in a ratio of 0.596(3)/0.404(3), MQOAHM (a) and MQOAHM (b), respectively. MQOAHM was characterized by means of various spectroscopic tools ESI-MS, IR, 1H &13C NMR analyses. Density Functional Theory (DFT) method, B3LYP, 6-311++G(d,p) basis set was used to optimize MQOAHM molecule. The obtained theoretical structure and experimental structure were superimposed on each other, and the correlation between them was calculated. The Highest Occupied Molecular Orbital (HOMO) and Lowest Unoccupied Molecular Orbital (LUMO) were created, and the energy gap between these orbitals was calculated. For analyzing intermolecular interactions, Molecular Electrostatic Potential (MEP) and Hirshfeld Surface Analysis were studied. For a fair comparative study, the two forms of the title compound were docked together with 18 approved drugs and N3 under precisely the same conditions. The disordered molecule structure's binding scores against 7BQY were -7.0 and -6.9 kcal/mol-1 for MQOAHM (a) and MQOAHM (b), respectively. Both the forms show almost identical superimposed structures and scores indicating that the disorder of the molecule, in this study, has no obvious effect. The high binding score of the molecule was attributed to the multi-hydrogen bond and hydrophobic interactions between the ligand and the receptor's active amino acid residues. Worth pointing out here that the aim of using the free energy in Silico molecular docking approach is to rank the title molecule compared to the wide range of approved drugs and a well-established ligand N3. The binding scores of all the molecules used in this study are ranged from -9.9 to -4.5 kcal/mol-1. These results and the supporting statistical analyses suggest that this malonate-based ligand merits further research in the context of possible therapeutic agents for COVID-19. Cheap computational techniques, PASS, Way2drug and ADMET, online software tools, were used in this study to uncover the title compound's potential biological activities and cytotoxicity.
RESUMO
Synthesis of a new fluorinated nucleoside of 6,7-difluoro-2-methyl-4-quinazolinone was described. 2-Amino-4,5-difluorobenzoic acid 1 reacts with (CH3CO)2O followed by ammonia to form (1H)-6,7-difluoro-2-methyl-4-quinazolinone 3a. Ribosylation of a silylated 4 with l-O-acety1-2,3-5-tri-O-benzoyl-α-D-ribofuranose 5 forms a protected nucleoside 6 then unprotected from 6 to give a free nucleoside 7. Greener pasture biological docking of the cystine protease of COVID-19 [Mpro, code 7BQY, PDB] by novel nucleoside and fluoroquinazoline compounds is presented. LIGPLOT (2D) representations calculated for the same ligands are shown. A superposition of remdesivir approved medicine, N3 inhibitor, and our ligands docked together into the binding protein of 7BQY is also given for a fair comparison. The binding affinities of remdesivir, N3 inhibitor, the nucleoside 7, and fluoroquinazoline 3a, 3b compounds with 7BQY calculated under the same conditions are -7.7, -7.4, -7.6, -6.1, and -6.1 kcal mol-1, respectively. The high values were due to the existence of many hydrophobic interactions and hydrogen bonds between the ligands and the active amino acid residues of the receptor, indicating a promising candidate as a COVID-19 inhibitor. Pro Tox -II server showed that compound 7 has a similar feature to the approved antiviral drug remdesivir for COVID-19. Additionally, a fascinating molecular modeling investigation showed that our nucleoside demonstrated good binding inhibition of AChE enzyme towards advancing an efficient medication against Alzheimer's disease. Finally, DFT has been conducted to illustrate the MD results in terms of the molecular descriptor-based structural activity relationship calculated from FMOs.
RESUMO
Schiff bases encompassing a 1,2,3-triazole motif were synthesized using an efficient multi-step synthesis. The formations of targeted Schiff base ligands were confirmed by different spectroscopic techniques (FT-IR, 1H NMR, 13C NMR, and CHN analysis). The spectral data analysis revealed that the newly designed hydrazones exist as a mixture of trans-E and cis-E diastereomers. Densityfunctional theory calculations (DFT) for the Schiff bases showed that the trans-trans form has the lowest energy structure with maximum stability compared to the other possible geometrical isomers that could be present due to the orientation of the amidic NH-C=O group. The energy differences between the trans-trans on one side and syn-syn and syn-trans isomers on the other side were 9.26 and 5.56 kcal/mol, respectively. A quantitative structure-activity relationship investigation was also performed in terms of density functional theory. The binding affinities of the newly synthesized bases are, maybe, attributed to the presence of hydrogen bonds together with many hydrophobic interactions between the ligands and the active amino acid residue of the receptor. The superposition of the inhibitor N3 and an example ligand into the binding pocket of 7BQY is also presented. Further interesting comparative docking analyses were performed. Quantitative structure-activity relationship calculations are presented, illustrating possible inhibitory activity. Further computer-aided cytotoxicity analysis by Drug2Way and PASS online software was carried out for Schiff base ligands against various cancer cell lines. Overall, the results of this study suggest that these Schiff base derivatives may be considered for further investigation as possible therapeutic agents for COVID-19.
RESUMO
The highly contagious nature of Covid-19 attracted us to this challenging area of research, mainly because the disease is spreading very fast and until now, no effective method of a safe treatment or a vaccine is developed. A library of novel 1,2,3-triazoles based 1,2,4-triazole, 1,3,4-oxadiazole and/or 1,3,4-thiadiazole scaffolds were designed and successfully synthesized. Different spectroscopic tools efficiently characterized all the newly synthesized hybrid molecules. An interesting finding is that some of the newly designed compounds revealed two isomeric forms. The ratio is affected by the size of the attached group as well as the type of the heteroatom forming the side ring attached to the central 1,2,3-triazole ring. The experimental spectroscopic data is in agreement with the DFT calculations at B3LYP 6-31G (d,p) with regard to the geometrical conformation of the prepared compounds. The DFT results revealed that the stability of one isomeric form over the other in the range of 0.057-0.161â Kcal mol-1. A docking study was performed using PyRx and AutoDockVina to investigate the activity of the prepared 1,2,3-triazoles as antiviral agents. Bond affinity scores of the 1,2,3-triazole derivatives were detected in the range of -6.0 to -8.8â kcal/mol showing binding to the active sites of the 6LU7 protease and hence could be anticipated to inhibit the activity of the enzyme. Verification of the docking results was performed using the Mpro alignment of coronaviruses substrate-binding pockets of COVID-19 against the ligands. As per these results, it can be proposed that the title hybrid molecules are acceptable candidates against COVID-19 for possible medicinal agents.
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Promoting the use of agricultural waste is one of the newly prepared water and environment friendly agriculture strategies in the Kingdom of Saudi Arabia (KSA). The objective of this research was to study the efficiency of cultivating oyster mushroom (Pleurotus ostreatus) on date palm wastes mixed with other agricultural wastes available in KSA. Four agricultural wastes were mixed with date palm leaves at different ratios, with two supplements and three spawn rates were used. Wheat straw mixed with date palm at ratio of 25 (date palm): 75 (agro-waste) showed the best results in most of the parameters measured. Corn meal was superior over wheat bran as a supplement in all treatments. Parameter values increased with the increase of the spawn rate of P. ostreatus. Treatments with date palm leave wastes contained higher carbohydrates and fibers. No significant differences were found among the fruiting bodies produced on the different agro-wastes studied for the different proximates analyzed. Analyses of metal concentration showed that potassium was the highest in all the treatments tested followed by Na, Mg, Ca, and Zn. This is the first study that reported the success of growing oyster mushroom on date palm leaf wastes mixed with other agro-wastes obtainable in KSA.
RESUMO
Reduced height (Rht)-1 and Photoperiod (Ppd) have major effects on the adaptability of bread wheat (Triticum aestivum) to specific environments. Ppd-D1a is a photoperiod insensitive allele that reduces time to flowering. The gibberellin (GA) insensitive alleles Rht-B1b and Rht-D1b shorten plant stature and were important components of the 'green revolution'. Two additional Rht-B1 alleles were recently identified that contain a 160 or 197 bp insertion upstream of the coding region and may affect plant height or GA sensitivity Wilhelm et al. (Theor Appl Gen doi: 10.1007/s00122-013-2088-7 , 2013b). We determined the frequency of the five alleles in a worldwide core collection of 372 wheat accessions (372CC) and estimated their effects on height, days to heading, and GA sensitivity when the collection was grown in pots outdoors or in the glasshouse. This revealed that each allele was widespread geographically with frequencies ranging from 0.12 to 0.25. Ppd-D1a was associated with significant (p ≤ 0.05) reductions in days to heading and height relative to photoperiod sensitive Ppd-D1b. Relative to wild type, Rht-B1b and Rht-D1b each resulted in significant reductions in height (approximately 30 %) and GA sensitivity. The 160 and 197 bp alleles were associated with significant height reductions of 18 and 12 %, respectively, and with non-significant reductions in GA sensitivity relative to wild type. Two statistical methods were developed and used to estimate GA sensitivity of the 372CC accessions, but novel GA insensitive alleles were not identified. Further characterization of the Rht-B1 insertion alleles is required, but our results suggest these may enable fine adjustments in plant height.
Assuntos
Genes de Plantas , Giberelinas/metabolismo , Fotoperíodo , Proteínas de Plantas/genética , Triticum/genética , Alelos , Pão , Frequência do Gene , Loci Gênicos , Genótipo , Fenótipo , Proteínas de Plantas/metabolismo , Análise de Sequência de DNA , Triticum/crescimento & desenvolvimentoRESUMO
The introgression of Reduced height (Rht)-B1b and Rht-D1b into bread wheat (Triticum aestivum) varieties beginning in the 1960s led to improved lodging resistance and yield, providing a major contribution to the 'green revolution'. Although wheat Rht-1 and surrounding sequence is available, the genetic composition of this region has not been examined in a homoeologous series. To determine this, three Rht-1-containing bacterial artificial chromosome (BAC) sequences derived from the A, B, and D genomes of the bread wheat variety Chinese Spring (CS) were fully assembled and analyzed. This revealed that Rht-1 and two upstream genes were highly conserved among the homoeologs. In contrast, transposable elements (TEs) were not conserved among homoeologs with the exception of intronic miniature inverted-repeat TEs (MITEs). In relation to the Triticum urartu ancestral line, CS-A genic sequences were highly conserved and several colinear TEs were present. Comparative analysis of the CS wheat BAC sequences with assembled Poaceae genomes showed gene synteny and amino acid sequences were well preserved. Further 5' and 3' of the wheat BAC sequences, a high degree of gene colinearity is present among the assembled Poaceae genomes. In the 20 kb of sequence flanking Rht-1, five conserved non-coding sequences (CNSs) were present among the CS wheat homoeologs and among all the Poaceae members examined. Rht-A1 was mapped to the long arm of chromosome 4 and three closely flanking genetic markers were identified. The tools developed herein will enable detailed studies of Rht-1 and linked genes that affect abiotic and biotic stress response in wheat.
Assuntos
Mapeamento Cromossômico , Cromossomos de Plantas/genética , Genes de Plantas/genética , Genoma de Planta/genética , Poaceae/genética , Triticum/genética , Cromossomos Artificiais Bacterianos , DNA de Plantas/genética , Marcadores Genéticos , Filogenia , Poaceae/classificação , Triticum/crescimento & desenvolvimentoRESUMO
The introduction of the Reduced height (Rht)-B1b and Rht-D1b semidwarfing genes led to impressive increases in wheat (Triticum aestivum) yields during the Green Revolution. The reduction in stem elongation in varieties containing these alleles is caused by a limited response to the phytohormone gibberellin (GA), resulting in improved resistance to stem lodging and yield benefits through an increase in grain number. Rht-B1 and Rht-D1 encode DELLA proteins, which act to repress GA-responsive growth, and their mutant alleles Rht-B1b and Rht-D1b are thought to confer dwarfism by producing more active forms of these growth repressors. While no semidwarfing alleles of Rht-A1 have been identified, we show that this gene is expressed at comparable levels to the other homeologs and represents a potential target for producing novel dwarfing alleles. In this study, we have characterized additional dwarfing mutations in Rht-B1 and Rht-D1. We show that the severe dwarfism conferred by Rht-B1c is caused by an intragenic insertion, which results in an in-frame 90-bp insertion in the transcript and a predicted 30-amino acid insertion within the highly conserved amino-terminal DELLA domain. In contrast, the extreme dwarfism of Rht-D1c is due to overexpression of the semidwarfing Rht-D1b allele, caused by an increase in gene copy number. We show also that the semidwarfing alleles Rht-B1d and Rht-B1e introduce premature stop codons within the amino-terminal coding region. Yeast two-hybrid assays indicate that these newly characterized mutations in Rht-B1 and Rht-D1 confer "GA-insensitive" dwarfism by producing DELLA proteins that do not bind the GA receptor GA INSENSITIVE DWARF1, potentially compromising their targeted degradation.
Assuntos
Giberelinas/farmacologia , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/genética , Triticum/crescimento & desenvolvimento , Triticum/genética , Alelos , Sequência de Aminoácidos , Sequência de Bases , DNA de Plantas/química , DNA de Plantas/genética , Dados de Sequência Molecular , Mutação , Fenótipo , Proteínas de Plantas/metabolismo , Caules de Planta/genética , Caules de Planta/crescimento & desenvolvimento , Poliploidia , RNA Mensageiro/genética , RNA de Plantas/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Triticum/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
During meiosis, chromosome numbers are halved, leading to haploid gametes, a process that is crucial for the maintenance of a stable genome through successive generations. The process for the accurate segregation of the homologues starts in pre-meiosis as each homologue is replicated and the respective products are held together as two sister chromatids via specific cohesion proteins. At the start of meiosis, each chromosome must recognise its homologue from amongst all the chromosomes present in the nucleus and then associate or pair with that homologue. This process of homologue recognition in meiosis is more complicated in polyploids because of the greater number of related chromosomes. Despite the presence of these related chromosomes, for polyploids such as wheat to produce viable gametes, they must behave as diploids during meiosis with only true homologues pairing. In this review, the relationship between the Ph1 cyclin-dependent kinase (CDK)-like genes in wheat and the CDK2 genes in mammals and their involvement in controlling this process at meiosis is examined.
Assuntos
Pareamento Cromossômico/genética , Meiose/genética , Poliploidia , Triticum/genética , Sequência de Aminoácidos , Loci Gênicos , Dados de Sequência Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genéticaRESUMO
The Ph1 locus in hexaploid wheat is responsible for restricting chromosome pairing at meiosis to true homologues by suppressing homoeologous pairing. Based on detailed modelling studies and predicted ability to form complexes with cyclin-A and cyclin-dependent kinase inhibitor such as p27, Triticum aestivum-5B2 (( Ta ) 5B2) is suggested to be a wheat analogue of human CDK2 enzyme. A blast analysis of the protein data bank using the amino acid sequence of the protein expressed by the 5B2 copy of the cdk-like cluster of genes at the Ph1 locus (( Ta ) 5B2) identified humans CDK2 as a top hit. In this analysis, the canonical cyclin binding motif PSTAIRE of CDK2 is replaced by a novel DARTLRE motif and Thr160 residue, phosphorylation of which is required for positive regulation of CDK2, is replaced by a tyrosine (Tyr174) in ( Ta ) 5B2. Despite these differences, detailed analyses show that all residues known to be important for cyclin binding are either fully conserved or whenever there is alteration in ( Ta ) 5B2, a corresponding but comparable alteration is also observed in plant cyclins notably cyclin-A of Arabidopsis thaliana. Moreover, the Thr160/Tyr174 substitution is also accommodated by suitable alterations in the 3D space around Tyr174 and the 3D model of ( Ta ) 5B2 predicts Tyr174 to play the same role as Thr160 plays in CDK2.
Assuntos
Quinase 2 Dependente de Ciclina/química , Quinase 2 Dependente de Ciclina/metabolismo , Loci Gênicos/genética , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Triticum/enzimologia , Triticum/genética , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Proteínas Inibidoras de Quinase Dependente de Ciclina/metabolismo , Ciclinas/química , Ciclinas/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Fosforilação , Fosfotreonina/metabolismo , Proteínas de Plantas/genética , Ligação Proteica , Estrutura Secundária de Proteína , Relação Estrutura-AtividadeRESUMO
A high-throughput two-step PCR strategy for the identification of selected genes from a BAC library derived from hexaploid wheat (16,974 Mbp) is described. The screen is based on the pooling of DNA from BAC clones into 675 "superpools" arrayed in a three-dimensional configuration. Each BAC clone is represented in three superpools to allow the identification of candidate 384-well plates of clones after the first round of PCR; identification is facilitated by an associated Perl script. A second round of PCR detects the specific BAC clone within the candidate plate that corresponds to the gene of interest. Thus, a single copy of the target gene can be identified from the library of over 700,000 clones (approximately 5 genome equivalents) by assaying only three 384-well plates. The pooling strategy was validated by screening the library with primers specific for the reduced height (Rht-1a) gene. Using relatively stringent selection criteria, 13 Rht-containing clones were identified from 17 candidate plates, and sequence analysis of the amplified products showed that all three Rht homoeologues were represented. Furthermore, the method confirmed the estimated coverage of the BAC library. Thus, this methodology allows the rapid and cost-effective identification of genes, and their homoeologues, from large-insert libraries of complex genomes such as hexaploid wheat.
Assuntos
Cromossomos Artificiais Bacterianos/genética , Clonagem Molecular/métodos , Genes de Plantas/genética , Reação em Cadeia da Polimerase/métodos , Triticum/genética , Sequência de Bases , DNA de Plantas/química , DNA de Plantas/genética , Biblioteca Genômica , Dados de Sequência Molecular , Poliploidia , Reprodutibilidade dos Testes , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
BACKGROUND AND AIMS: Understanding Ph1, a dominant homoeologous chromosome pairing suppressor locus on the long arm of chromosome 5B in wheat Triticum aestivum L., is the core of the investigation in this article. The Ph1 locus restricts chromosome pairing and recombination at meiosis to true homologues. The importance of wheat as a crop and the need to exploit its wild relatives as donors for economically important traits in wheat breeding programmes is the main drive to uncover the mechanism of the Ph1 locus and regulate its activity. METHODS: Following the molecular genetic characterization of the Ph1 locus, five additional deletion mutants covering the region have been identified. In addition, more bacterial artificial chromosomes (BACs) were sequenced and analysed to elucidate the complexity of this locus. A semi-quantitative RT-PCR was used to compare the expression profiles of different genes in the 5B region containing the Ph1 locus with their homoeologues on 5A and 5D. PCR products were cloned and sequenced to identify the gene from which they were derived. KEY RESULTS: Deletion mutants and expression profiling of genes in the region containing the Ph1 locus on 5B has further restricted Ph1 to a cluster of cdk-like genes. Bioinformatic analysis of the cdk-like genes revealed their close homology to the checkpoint kinase Cdk2 from humans. Cdk2 is involved in the initiation of replication and is required in early meiosis. Expression profiling has revealed that the cdk-like gene cluster is unique within the region analysed on 5B in that these genes are transcribed. Deletion of the cdk-like locus on 5B results in activation of transcription of functional cdk-like copies on 5A and 5D. Thus the cdk locus on 5B is dominant to those on 5A and 5D in determining the overall activity, which will be dependent on a complex interplay between transcription from non-functional and functional cdk-like genes. CONCLUSIONS: The Ph1 locus has been defined to a cdk-like gene cluster related to Cdk2 in humans, a master checkpoint gene involved in the initiation of replication and required for early meiosis.