Discovery of gene families and alternatively spliced variants by RecA-mediated cloning

Probing the functional complexity of the human genome will require new gene cloning techniques, not only to discover intraspecies gene homologs and interspecies gene orthologs, but also to identify alternatively spliced gene variants. We report homologous cDNA cloning methods that allow cloning of gene family members, genes from different species, and alternatively spliced gene variants. We cloned human 14-3-3 gene family members using DNA probes with as much as 35% sequence divergence, cloned alternatively spliced gene forms of Rad51D, and cloned a novel splice form of the human 14-3-3 theta gene with a unique expression pattern. Interspecies gene cloning was demonstrated for the mouse Rad51C and mouse beta-actin genes using human gene probes. The gene family cloning method is fast, efficient, and free from PCR errors; moreover, it exploits the abilities of RecA protein to pair homologous or partially homologous DNA sequences stably in kinetically trapped, multistranded DNA hybrids that can be used for subsequent gene clone enrichment.     

比格犬一种新雌激素β受体可变剪切体的克隆及鉴定

目的对比格犬雌激素β受体的新剪切体进行克隆与鉴定。方法以比格犬垂体组织为模板,用RT-PCR方法从垂体组织中扩增雌激素β受体,电泳确定新剪切体的存在情况.并对其进行克隆和序列测定.结果利用设计的引物得到2条明显电泳条带,通过测序表明其中一种为新的可变剪切体,目前此可变剪切体尚未见报道。结论得到了雌激素β受体的一种新的可变剪切体,有助于研究雌激素β受体可变剪切体的功能及其在生殖调控过程中的作用。     

Alternative non-coding exons support serotonin transporter mRNA expression in the brain and gut

A number of studies in recent years have linked polymorphisms within the serotonin transporter (5HTT) gene to affective disorders and anxiety traits. The human 5HTT mRNA is alternatively spliced, and the splice variants are equally expressed in the human placental cell line and dorsal raphe. In this study, using 5' rapid amplification of cDNA ends, we show that the rat 5HTT mRNA is alternatively spliced, leading to three distinct mRNAs differing in the 5' untranslated region. To determine whether the three alternatively spliced mRNA species that contain one of the following untranslated regions (i) exon 1A, 63 bp (ii) exon 1A + 1B, 125 bp or (iii) exon 1C, 101 bp, were expressed in a tissue-specific manner, we used RT-PCR and exon-specific oligonucleotide hybridization. Our results suggest two of the variants (1A + 1B and 1A) may utilize the same promoter; however, they are not equally expressed. While in the adult CNS and adrenal medulla, the shorter mRNA consisting of exon 1A was considerably more abundant, in the stomach and heart, the two variants were equally expressed. The third splice variant exon 1C is only expressed in the gut and to a lesser extent in the heart. The data from this study suggest the splice variant consisting of exon 1C may utilize a distinct promoter compared to the other two.     

Primer design versus PCR bias in methylation independent PCR amplifications

Many protocols in methylation studies utilize one primer set to generate a PCR product from bisulfite modified template regardless of its methylation status (methylation independent amplification MIP). However, proportional amplification of methylated and unmethylated alleles is hard to achieve due to PCR bias favoring amplification of unmethylated relatively GC poor sequence. Two primer design systems have been proposed to overcome PCR bias in methylation independent amplifications. The first advises against including any CpG dinucleoteides into the primer sequence (CpG-free primers) and the second, recently published by us, is based on inclusion of a limited number of CpG sites into the primer sequence. Here we used the Methylation Sensitive High Resolution Melting (MS-HRM) technology to investigate the ability of primers designed according to both of the above mentioned primer design systems to proportionally amplify methylated and unmethylated templates. Ten “CpG-free” primer pairs and twenty primers containing limited number of CpGs were tested. In reconstruction experiments the “CpG-free” primers showed primer specific sensitivity and allowed us to detect methylation levels in the range from 5 to 50%. Whereas while using primers containing limited number of CpG sites we were able to consistently detect 1–0.1% methylation levels and effectively control PCR amplification bias. In conclusion, the primers with limited number of CpG sites are able to effectively reverse PCR bias and therefore detect methylated templates with significantly higher sensitivity than CpG free primers.     

EasyExonPrimer

EasyExonPrimer is a web-based software that automates the design of PCR primers to amplify exon sequences from genomic DNA. EasyExonPrimer is written in Perl and uses Primer3 to design PCR primers based on the genome builds and annotation databases available at the University of California, Santa Cruz (UCSC) Genome Browser database (http://genome.ucsc.edu/). It masks repeats and known single nucleotide polymorphism (SNP) sites in the genome and designs standardised primers using optimised conditions. Users can input genes by RefSeq mRNA ID, gene name or keyword. The primer design is optimised for large-scale resequencing of exons. For exons larger than 1 kb, the user has the option of breaking the exon sequence down into overlapping smaller fragments. All primer pairs are then verified using the In-Silico PCR software to test for uniqueness in the genome. We have designed >1000 pairs of primers for 90 genes; 95% of the primer pairs successfully amplified exon sequences under standard PCR conditions without requiring further optimisation. AVAILABILITY: EasyExonPrimer is available from http://129.43.22.27/~primer/. The source code is also available upon request. CONTACT: Xiaolin Wu (forestwu@mail.nih.gov).     

Characterizing homologues of crop domestication genes in poorly described wild relatives by high‐throughput sequencing of whole genomes

Wild crop relatives represent a source of novel alleles for crop genetic improvement. Screening biodiversity for useful or diverse gene homologues has often been based upon the amplification of targeted genes using available sequence information to design primers that amplify the target gene region across species. The crucial requirement of this approach is the presence of sequences with sufficient conservation across species to allow for the design of universal primers. This approach is often not successful with diverse organisms or highly variable genes. Massively parallel sequencing (MPS) can quickly produce large amounts of sequence data and provides a viable option for characterizing homologues of known genes in poorly described genomes. MPS of genomic DNA was used to obtain species‐specific sequence information for 18 rice genes related to domestication characteristics in a wild relative of rice, Microlaena stipoides. Species‐specific primers were available for 16 genes compared with 12 genes using the universal primer method. The use of species‐specific primers had the potential to cover 92% of the sequence of these genes, while traditional universal primers could only be designed to cover 80%. A total of 24 species‐specific primer pairs were used to amplify gene homologues, and 11 primer pairs were successful in capturing six gene homologues. The 23 million, 36‐base pair (bp) paired end reads, equated to an average of 2X genome coverage, facilitated the successful amplification and sequencing of six target gene homologues, illustrating an important approach to the discovery of useful genes in wild crop relatives.     

A class III myosin expressed in the retina is a potential candidate for Bardet-Biedl syndrome

Class III myosins are actin-based motors with amino-terminal kinase domains. Expression of these motors is highly enhanced in retinal photoreceptors. As mutations in the gene encoding NINAC, a Drosophila melanogaster class III myosin, cause retinal degeneration, human homologs of this gene are potential candidates for human retinal disease. We have recently reported the cloning of MYO3A, a human myosin III expressed predominantly in the retina and retinal pigmented epithelium [1]. The map locus of MYO3A is close to, but does not overlap, that of human Usher's 1F [2]. Here we introduce a shorter class III myosin isoform, MYO3B, which is expressed in the retina, kidney, and testis. We describe the cDNA sequence, genomic organization, and splice variants of MYO3B expressed in the human retina. A product of 36 exons, MYO3B has several splice variants containing either one or two calmodulin binding (IQ) motifs in the neck domain and one of three predominant tail variations: a short tail ending just past the second IQ motif, or two alternatively spliced longer tails. MYO3B maps to 2q31.1-q31.2, a region that overlaps the locus for a Bardet-Biedl syndrome (BBS5) linked to markers at 2q31 [3].     

The human XPG gene: gene architecture, alternative splicing and single nucleotide polymorphisms

Defects in the XPG DNA repair endonuclease gene can result in the cancer-prone disorders xeroderma pigmentosum (XP) or the XP-Cockayne syndrome complex. While the XPG cDNA sequence was known, determination of the genomic sequence was required to understand its different functions. In cells from normal donors, we found that the genomic sequence of the human XPG gene spans 30 kb, contains 15 exons that range from 61 to 1074 bp and 14 introns that range from 250 to 5763 bp. Analysis of the splice donor and acceptor sites using an information theory-based approach revealed three splice sites with low information content, which are components of the minor (U12) spliceosome. We identified six alternatively spliced XPG mRNA isoforms in cells from normal donors and from XPG patients: partial deletion of exon 8, partial retention of intron 8, two with alternative exons (in introns 1 and 6) and two that retained complete introns (introns 3 and 9). The amount of alternatively spliced XPG mRNA isoforms varied in different tissues. Most alternative splice donor and acceptor sites had a relatively high information content, but one has the U12 spliceosome sequence. A single nucleotide polymorphism has allele frequencies of 0.74 for 3507G and 0.26 for 3507C in 91 donors. The human XPG gene contains multiple splice sites with low information content in association with multiple alternatively spliced isoforms of XPG mRNA.     

Don't put all your eggs in one basket: a cost‐effective and powerful method to optimize primer choice for rRNA environmental community analyses using the Fluidigm Access Array

With the increasing democratization of high‐throughput sequencing (HTS) technologies, along with the concomitant increase in sequence yield per dollar, many researchers are exploring HTS for microbial community ecology. Many elements of experimental design can drastically affect the final observed community structure, notably the choice of primers for amplification prior to sequencing. Some targeted microbes can fail to amplify due to primer‐targeted sequence divergence and be omitted from obtained sequences, leading to differences among primer pairs in the sequenced organisms even when targeting the same community. This potential source of taxonomic bias in HTS makes it prudent to investigate how primer choice will affect the sequenced community prior to investing in a costly community‐wide sequencing effort. Here, we use Fluidigm's microfluidic Access Arrays (IFC) followed by Illumina^® MiSeq Nano sequencing on a culture‐derived local mock community to demonstrate how this approach allows for a low‐cost combinatorial investigation of primer pairs and experimental samples (up to 48 primer pairs and 48 samples) to determine the most effective primers that maximize obtained communities whilst minimizing taxonomic biases.     

肝素酶基因可变剪接体的克隆及鉴定

目的:克隆肝素酶基因的可变剪接体并测序。方法:根据人肝素酶的cDNA序列设计引物,用RT-PCR方法从正常人外周血白细胞中扩增肝素酶基因的可变剪接体,构建至pGEM-T Easy载体中,转化大肠杆菌DH5α感受态细胞,筛选阳性克隆并进行序列测定。结果:获得了肝素酶基因的3种可变剪接体形式,即5号外显子缺失可变剪接体、6号外显子缺失可变剪接体、5和6号外显子缺失可变剪接体,其中后2种可变剪接体尚未报道。结论:克隆了肝素酶基因的3种可变剪接体,有助于研究各种肝素酶可变剪接体编码蛋白的结构和功能及其在肿瘤发生转移过程中的作用。