Quantitative trait locus mapping using sets of near-isogenic lines: relative power comparisons and technical considerations

 The power to detect QTL using near-isogenic line (NIL) comparisons versus recombinant inbred (RI) populations was assessed. The power to detect QTL was found to be generally greater when using RI populations than when using NIL contrasts. Power to detect QTL with NIL contrasts never exceeded that of RI populations when the number of RI lines is maximized relative to replication of lines for a given number of experimental units. The relative power of NIL contrasts is highest for traits with high heritability and when a gain in precision is realized due to increased replication of entries. Although NIL populations are generally less powerful than RI populations of similar size, some practical considerations may enhance the value of these materials. Availability of NILs allows the genetic effect of a specific chromosome region to be determined by comparing two lines; all RI lines in a population need to be rescored for each new trait even if the effect of a specific chromosome region is suspected. NIL comparisons may allow genetic differences to be detected by visual inspection; genetic effects can only be expressed as means and variances with recombinant inbred populations. In summary, RI populations generally, and in some cases, substantially, provide better power for QTL detection than NIL comparisons. Practical considerations, however, indicate that many factors need to be considered when choosing a population structure to meet an experimental objective. Received: 12 December 1996 / Accepted: 21 March 1997     

Characterization of newly established colorectal cancer cell lines: correlation between cytogenetic abnormalities and allelic deletions associated with multistep tumorigenesis

We have established a series of 20 colorectal cancer cell lines and performed cytogenetic and RFLP analyses to show that the recurrent genetic abnormalities of chromosomes 1, 5, 17 and 18 associated with multistep tumorigenesis in colorectal cancer, and frequently detected as recurrent abnormalities in primary tumours, are also retained in long-term established cell lines. Earlier studies by us and other investigators showed that allelic losses of chromosomes 1 and 17 in primary colorectal cancers predicted poorer survival for the patients (P = 0.03). We utilized the cell lines to identify specific chromosomal sites or gene(s) on chromosomes 1 and 17 which confer more aggressive phenotype. Cytogenetic deletions of chromosome 1p were detected in 14 out of the 20 (70%) cell lines, whereas allelic deletions for 1p using polymorphic markers were detected in 13 out of 18 (72%) informative cell lines for at least one polymorphic marker. We have performed Northern blotting, immunohistochemical staining (p53 mRNA, protein) and RFLP analysis using several probes including p53 and nm23. RFLP analysis using a total of seven polymorphic markers located on 17p and 17q arms showed allelic losses aroundthe p53 locus in 16 out of the 20 cell lines (80%), four of which were losses of thep53 locus itself. In addition, seven cell lines (out of nine informative cases) also showed losses of thenm23 gene, four with concurrent losses of thep53 locus, while the remaining three were homozygous. In addition, five out of seven cell lines withnm23 deletions were derived from hepatic metastatic tumours, and one cell line was obtained from recurrent tumour. A comparison between allelic deletions of 1p and functional loss ofnm23 gene revealed a close association between these two events in cell lines derived from hepatic metastasis. Following immunohistochemical staining, nine out of the twenty cell lines showed high levels (25–80%) of mutant p53, four showed intermediate levels (>20%), and seven had undetectable levels of the protein. Of these seven, four showed complete absence of mRNA. Of the remaining three cell lines one showed aberrant mRNA due to germline rearrangement of thep53 gene, whereas in two cell lines normal levels of mRNA were present. Nineteen of the 20 cell lines had normal germline configurations for thep53 gene, while one showed a rearrangement. These data suggest that functional loss ofp53 andnm23 genes accomplished by a variety of mechanisms may be associated with poor prognosis and survival. In addition, concurrent deletions of chromosome regions 17p, 17q and 1p were closely associated with high-stage hepatic metastatic disease. These cell lines with well-characterized genetic alterations and known clinical history provide an invaluable source of material for various biological and clinical studies relating to multistep colorectal tumorigenesis.     

Lethal bithorax complex mutations of Drosophila melanogaster show no germ line maternal effects

Three Ultrabithorax (Ubx) alleles and three different deficiencies of the bithorax complex (BX-C) of Drosophila melanogaster have been tested for maternal effects in the germ line. The dominant female sterile technique was used. The Ubx alleles and a deletion of the abdominal region of the BX-C are homozygous viable in germ line clones and show no maternal effects. Two deletions which lack the proximal portion of the BX-C are lethal in the female germ line indicating either that these deficiencies lack genes apart from the BX-C that are necessary for fertility or that there are BX-C genes that are essential for normal maternal germ line function. The significance of the bias in the isolation of only zygotic mutations at the BX-C are discussed with respect to these results.     

Molecular cytogenetic characterization of Roegneria ciliaris chromosome additions in common wheat

The development of alien addition lines is important both for transferring useful genes from related species into common wheat and for studying the relationship between alien chromosomes and those of wheat. Roegneria ciliaris (2n=4x=28, S^cS^cY^cY^c) is reported to be a potential source of resistance to wheat scab, which may be useful in wheat improvement. The amphiploid common wheat-R. ciliaris and BC₁F₇ or BC₂F₆ derivatives were screened by C-banding, genomic in situ hybridization (GISH), fluorescent in situ hybridization (FISH) and restriction fragment length polymorphism (RFLP) for the presence of R. ciliaris chromatin introgressed into wheat. Six lines were identified as disomic chromosome additions (DA), one as a ditelosomic addition (Dt), two as double disomic additions (dDA) and one as a monosomic chromosome addition (MA). RFLP analysis using wheat homoeologous group-specific clones indicated that the R. ciliaris chromosomes involved in these lines belong to groups 1, 2, 3, 5 and 7. The genomic affinities of the added R. ciliaris chromosomes were determined by FISH analysis using the repetitive sequence pCbTaq4.14 as a probe. These data suggest that the R. ciliaris chromosomes in five lines belong to the S^c genome. Based on the molecular cytogenetic data, the lines are designated as DA2S^c#1, Dt2S^c#1L, DA3S^c#1, dDA1S^c#2+5Y^c#1, DA5Y^c#1, DA7S^c#1, DA7Y^c#1 and MA?Y^c#1. Based on the present and previous work, 8 of the 14 chromosomes of R. ciliaris have been transferred into wheat.     

鼻咽癌细胞系与永生化的鼻咽上皮细胞系蛋白质表达差异分析

鼻咽癌对我国南部居民的健康造成严重的威胁.为了研究鼻咽癌的发病机理，本研究采用了蛋白质组学技术分析和比较了鼻咽癌细胞系（HNE1和CNE1）与永生化的鼻咽上皮细胞系的蛋白质表达谱.采用双向凝胶电泳分离提取的全细胞蛋白质，通过PDQuest软件分析找出在肿瘤中表达变化的蛋白质点，用基质辅助激光解析电离飞行时间串联质谱(MALDI- TOF/TOF-MS)进行鉴定.共得到了15个在肿瘤细胞系中表达上调和18个在肿瘤细胞系中表达下调的蛋白质，并对其中一些蛋白质的表达进行免疫印迹的验证.这些表达差异的蛋白质与细胞的增殖和调亡、癌症的转移，细胞骨架，信号传导等有关.本研究鉴定了一批可能作为鼻咽癌治疗的药物靶标的蛋白质，并对研究鼻咽癌发病机理提供了相关的线索.     

Simple But Efficacious Enrichment of Integral Membrane Proteins and Their Interactions for In-Depth Membrane Proteomics

Membrane proteins play essential roles in various cellular processes, such as nutrient transport, bioenergetic processes, cell adhesion, and signal transduction. Proteomics is one of the key approaches to exploring membrane proteins comprehensively. Bottom–up proteomics using LC–MS/MS has been widely used in membrane proteomics. However, the low abundance and hydrophobic features of membrane proteins, especially integral membrane proteins, make it difficult to handle the proteins and are the bottleneck for identification by LC–MS/MS. Herein, to improve the identification and quantification of membrane proteins, we have stepwisely evaluated methods of membrane enrichment for the sample preparation. The enrichment methods of membranes consisted of precipitation by ultracentrifugation and treatment by urea or alkaline solutions. The best enrichment method in the study, washing with urea after isolation of the membranes, resulted in the identification of almost twice as many membrane proteins compared with samples without the enrichment. Notably, the method significantly enhances the identified numbers of multispanning transmembrane proteins, such as solute carrier transporters, ABC transporters, and G-protein–coupled receptors, by almost sixfold. Using this method, we revealed the profiles of amino acid transport systems with the validation by functional assays and found more protein–protein interactions, including membrane protein complexes and clusters. Our protocol uses standard procedures in biochemistry, but the method was efficient for the in-depth analysis of membrane proteome in a wide range of samples.