Intragenic and intergenic sequences regulating the expression of the 5'-to-5' linked adult α-and β-globin genes from large yellow croaker Pseudosciaena crocea

One adult α-globin gene and one β-globin gene have been cloned from the large yellow croaker Pseudosciaena crocea. Linkage analysis indicated that the α- and β-globin genes were oriented head-to-head relative to each other. To identify the regulatory elements present in the intergenic and intragenic regions of the globin complex, the intergenic region alone or together with the β-globin gene first intron was cloned into the luciferase-reporter vector pGL3-Basic respectively, and the chimeric constructs were tran- siently transfected into Vero cells and primary fish erythrocytes. The intergenic region cannot support the high-level expression of luciferase. However, the promoter activity of the intergenic region was strongly stimulated by the positive regulatory elements （PRE） located in the β-globin gene intron 1. Thus, it is proposed that the intergenic promoters and intragenic PRE were necessary for the effective expression of the linked α- and β-globin genes.

UHRF1/DNMT1—MZF1 axis-modulated intragenic site-specific CpGI methylation confers divergent expression and opposing functions of PRSS3 isoforms in lung cancer

As confusion mounts over RNA isoforms involved in phenotypic plasticity,aberrant CpG methylation-mediated disruption of alternative splicing is increasingly recognized as a driver of intratumor heterogeneity(ITH).Protease serine 3(PRSS3),possessing four splice variants(PRSS3-SVs;PRSS3-V1—V4),is an indispensable trypsin that shows paradoxical effects on cancer development.Here,we found that PRSS3 transcripts and their isoforms were divergently expressed in lung cancer,exhibiting opposing functions and clinical outcomes,namely,oncogenic PRSS3-V1 and PRSS3-V2 versus tumorsuppressive PRSS3-V3,by targeting different downstream genes.We identified an intragenic CpG island(iCpGI)in PRSS3.Hypermethylation of iCpGI was mediated by UHRF1/DNMT1 complex interference with the binding of myeloid zinc finger 1(MZF1)to regulate PRSS3 transcription.The garlic-derived compound diallyl trisulfide cooperated with 5-aza-2'-deoxycytidine to exert antitumor effects in lung adenocarcinoma cells through site-specific iCpGI demethylation specifically allowing MZF1 to upregulate PRSS3-V3 expression.Epigenetic silencing of PRSS3-V3 via i CpGI methylation(iCpGIm)in BALF and tumor tissues was associated with early clinical progression in patients with lung cancer but not in those with squamous cell carcinoma or inflammatory disease.Thus,UHRF1/DNMT1—MZF1 axismodulated site-specific iCpGIm regulates divergent expression of PRSS3-SVs,conferring nongenetic functional ITH,with implications for early detection of lung cancer and targeted therapies.

Ideal type 1 is caused by a point mutation in the a-tubulin gene that affects microtubule arrangement in soybean

Plant architecture is a target of crop improvement.The soybean mutant ideal type 1(it1)displays a pleiotropic phenotype characterized by compact plant architecture,reduced plant height,shortened petioles,wrinkled leaves,and indented seeds.Genetic analysis revealed that the pleiotropic phenotype was controlled by an incomplete dominant gene.We characterized the cellular phenotypes of it1 and positionally cloned the it1 locus.Detailed morphogenetic analysis of the it1 mutant revealed an excess of xylem cells and expanded phloem,and polygonal pavement cells.Positional cloning showed that the phenotype was caused by a G-to-A mutation in the second exon of the a-tubulin gene(Glyma.05G157300).The mutation altered microtubule arrangement in pavement cells,changing their morphology.Overexpression of Gmit1 resulted in an it1-like phenotype and polygonal pavement cells and microtubules of overexpressors were parallel or slightly inclined.Five suppressor mutants able to suppress the phenotype of it1 were obtained by EMS mutagenesis in the it1 background.All these mutants carried an additional mutation in the it1 gene.These results suggest that the pleiotropic phenotype of it1 is caused by the mutation in the atubulin gene.

A genetic diversity assessment of starch quality traits in rice landraces from the Taihu basin,China

There are nearly 1 000 rice landrace varieties in the Taihu basin, China. To assess the genetic diversity of the rice, 24 intragenic molecular markers（representing 17 starch synthesis-related genes） were investigated in 115 Taihu basin rice landraces and 87 improved cultivars simultaneously. The results show that the average genetic diversity and polymorphism information content values of the landraces were higher than those of improved cultivars. In total, 41 and 39 allele combinations（of the 17 genes） were derived from the landraces and improved cultivars, respectively; only two identical allele combinations were found bet ween the two rice variety sources. Cluster analysis, based on the molecular markers, revealed that the rice varieties could be subdivided into five groups and, within these, the japonica improved rice and japonica landrace rice varieties were in two separate groups. According to the quality reference criteria to classify the rice into grades, some of the landraces were found to perform we ll, in terms of starch quality. For example, according to NY /T595-2002 criteria from the Ministry of Agriculture of China, 25 and 33 landraces reached grade 1, in terms of their apparent amylose content and gel consistency. Th e varieties that had outstanding quality could be used as breeding materials for rice quality breeding programs in the future. Our study is useful for future applications, such as genetic diversity studies, the protection of rice variety and improvment of rice quality in breeding programs.

Diagnosis of Laron syndrome using monoplex-polymerase chain reaction technology with a whole-genome amplification template: A case report

BACKGROUND Laron syndrome(LS)is an autosomal recessive hereditary condition affecting only 1/1000000 births.The cause is associated with mutations in the growth hormone(GH)receptor(GHR),leading to GH insensitivity.LS patients typically present with severe growth retardation,obesity,and abnormal sexual maturation.Currently,LS diagnosis is performed post-delivery.Therefore,we assessed the efficiency of Pre-implantation Genetic Testing(PGT)coupled with monoplex-polymerase chain reaction(PCR)technology for detecting this monogenic disease in embryos from a couple confirmed as LS heterozygous carriers CASE SUMMARY The couple LS-carriers were confirmed by the presence of a first child born with LS.The couple underwent a standard in vitro fertilization(IVF)protocol.DNA was collected from trophectoderm cells from day 5 embryos.Whole genome amplification(WGA)was performed using a Sureplex DNA Amplification System and analyzed by PCR,targeting the deletion of the exons 5 and 6 in the GHR gene as well as PGT by Next-generation Sequencing(Illumina).Eleven embryos were collected and analyzed.27.3%were the wild type for GHR,45.5%were heterozygotes,and 18.2%homozygous mutants.One embryo yielded no results.Three 2-embryos transfers were performed;2 normal homozygous and four heterozygous carriers were selected for transfer.The first two transfers were unsuccessful,whereas the final transfer with two heterozygous embryos resulted in clinical pregnancy.The genomic composition of the fetus was verified,applying the same techniques using amniocytes,extracted after 21 wk of the ongoing pregnancy.The fetus was confirmed as GHR deletion in exon 5-6,carrier.A non-affected baby was born.CONCLUSION Here,we present a case demonstrating that using WGA as a template in addition to PCR targeting specific gene regions,exons 5 and 6 on the GHR gene,could identify LS carrier embryos.This provides evidence that WGA and PCR serve as an excellent tool to detect this specific monogenic disease in IVF embryos,thus allowing selection of candidate embryos for transfer successfully when a specific inherited genetic mutation/disease is suspected.

Evaluation on Genetic Quality and Traits of Rice Landraces in the Taihu Lake Area

In this paper,with 511 rice landraces in the Taihu Lake area as test materials,we select 19 starch synthesis-related intragenic molecular markers to detect the genetic quality of starch,and compare them with 86 bred varieties. The results show that the average polymorphic information content( PIC) of japonica landraces is 0. 1726,slightly higher than the average PIC( 0. 1101) of the bred japonica rice varieties.Based on Nei's genetic distance between materials,UPGMA method is used for clustering,and all study materials are divided into 6 groups.Group I mainly includes indica rice,the bred japonica rice varieties are mainly concentrated in the first half of Group II and Group III,and the japonica landraces are mainly concentrated in the second half of Group III,and Group Ⅳ,Ⅴ,Ⅵ. Both of them are in different regions,and there has been genetic differentiation. According to the national standard of high quality rice,it is found that many rice landraces in the Taihu Lake area have good quality and traits,and these varieties can be used for future high quality breeding.

The origin of Wx^(la) provides new insights into the improvement of grain quality in rice

Appearance and taste are important factors in rice(Oryza sativa) grain quality. Here, we investigated the taste scores and related eating-quality traits of533 diverse cultivars to assess the relationships between—and genetic basis of—rice taste and eating-quality. A genome-wide association study highlighted the Wx gene as the major factor underlying variation in taste and eating quality. Notably, a novel waxy(Wx) allele, Wx^(la), which combined two mutations from Wx^(b) and Wx^(in), exhibited a unique phenotype. Reduced GBSSI activity conferred Wx^(la) rice with both a transparent appearance and good eating quality. Haplotype analysis revealed that Wx^(la) was derived from intragenic recombination. In fact,the recombination rate at the Wx locus was estimated to be 3.34 kb/c M, which was about 75-fold higher than the genome-wide mean, indicating that intragenic recombination is a major force driving diversity at the Wx locus. Based on our results, we propose a new network for Wx evolution, noting that new Wx alleles could easily be generated by crossing genotypes with different Wx alleles. This study thus provides insights into the evolution of the Wx locus and facilitates molecular breeding for quality in rice.

Various infection status and molecular evidence for horizontal transmission and recombination of Wolbachia and Cardinium among rice planthoppers and related species

Wolbachia and Cardinium are widely distributed and are considered important for their ability to disturb reproduction and affect other fitness-related traits of their hosts. By using multilocus sequence typing （MLST）, RFLP （restriction fragment length poly- morphism） and 16S ribosomal DNA gene sequencing methods, we extensively surveyed Wolbachia and Cardinium infection status of four predominant rice planthoppers and one kind of leafhopper in different rice fields. The results demonstrated that Sogatellafurcifera （Horv^ith） and Laodelphax striatellus （Fallrn） were infected with the same Wolbachia strain （wStri）, while Nilaparvata lugens （St^l） and its closely related species Nilaparvata muiri China were infected with two phylogeneticlly distant strains, wLug and wMui, respectively. Three new Wolbachia strains （provisionally named wMfasl, wMfas2 and wMfas3） were detected in the leafhopper Macrostelesfascifrons （Stal）. Only S. furcifera was co-infected with Cardinium, which indicated that the distribution of Cardinium in these rice planthop- pers was narrower than that of Wolbachia. Unambiguous intragenic recombination events among these Wolbachia strains and incongruent phylogenetic relationships show that the connections between different Wolbachia strains and hosts were more complex than we expected. These results suggest that horizontal transmission and host associated special- ization are two factors affecting Wolbachia and Cardinium infections among planthoppers and their related species.