Rb1基因第16内含子内21个碱基缺失1例

目地研究双眼视网膜母细胞瘤患者Rb1基因杂合性突变的分子生物学特性。方法应用PCR—SSCP直接测序技术检测双眼视网膜母细胞瘤患者白细胞DNA中Rb1基因杂合性突变。结果50例证实有Rb1基因杂合性突变的病例中有1例发生于第16内含子中可以用3种定位方法解释、具有相同序列的21个碱基缺失。结论这种极为少见的Rb1基因突变方式可能是由于破坏了正常拼接位点的结构而激活了“隐蔽拼接位点”，导致异常的Rb1基因mRNA产生或由此影响整个拼接过程。     

Splicing of the mitochondrial group-II intron rl1: conserved intron-exon interactions diminish splicing efficiency

The mitochondrial intron rI1 is a self-splicing group-II intron of algal mitochondria that can be transferred into chloroplasts from the green alga Chlamydomonas reinhardtii for in vivo investigations (Herdenberger et al. 1994). Thus, rI1 is a suitable system to compare in vitro and in vivo RNA processing. Interestingly, rI1 shows correct RNA splicing, although typical cis-acting exon-sequences (IBS2, δ) of group-II introns are lacking. In order to examine the effect of these exon-intron interactions on splicing, we introduced the endogenous mitochondrial IBS2 sequence in order to produce optimal IBS2-EBS2 base pairing. In addition, the first nucleotide of the 3′exon (δ′) was substituted to create an optimal δ-δ′ interaction. Neither of the two mutations, nor a combination of both, had any effect on the precision of the splice-site selection. Unexpectedly, introduction of IBS2 led to a reduction in the efficiency of the second splicing step in vitro but not in vivo. These findings lead us to conclude that trans-acting factors are present in vivo to optimize splicing efficiency. The possibility is discussed that these factors may, for example, stabilize tertiary intron structures that are a prerequisite for correct RNA processing. Furthermore, our data indicate that similar trans-acting factors promote correct intron splicing in chloroplasts and mitochondria. Received: 18 October / 4 December 1997     

Expression of CD44V2 in transitional cell carcinoma of the urinary bladder and in urine

CD44 is the principal cell surface receptor for hyaluronate. Variant forms of the receptor, produced by alternative splicing, have been found to be associated with tumor progression in a variety of cancers. Based on investigations at the RNA level, it has recently been proposed that expression of CD44 variant V2 was present in urothelial cancer but not in normal urothelium. Since a distinctive marker for urothelial cancer would be extremely useful, frozen sections of normal urothelium and urothelial cancer were examined for expression of standard CD44 and CD44V2. Frozen sections of specimens of 35 patients with transitional cell carcinoma of the bladder, 16 specimens of normal bladder and 5 ureters were examined. Immunohistochemical staining was performed using a polyclonal antibody to CD44V2 (PAB CD44V2), a monoclonal antibody to CD44V2 (MAB CD44V2) and a monoclonal antibody to CD44S (MAB CD44S). CD44V2 and CD44S were also measured in lysates of urine sediments from 21 patients by enzyme-linked immunoabsorbent assay (ELISA). All investigated transitional cell carcinomas expressed CD44V2. There was no differentiation between invasive and noninvasive carcinoma. CD44V2 was also expressed in normal urothelium. Standard CD44 was expressed by the transitional cell carcinoma, normal urothelium, musculature and interstitial tissue. The amount of CD44V2 and CD44S in lysates of urine sediments is not correlated to diagnosis. In contrast to investigations at the RNA level, CD44V2 on the protein level seems not to be a distinctive marker for urothelial cancer. Therefore, CD44V2 will not be a useful diagnostic marker for detection of transitional cell carcinoma.     

Heterologous Expression of Rat Testis GABAA Receptor β3t Splicing Variant in CHO Cells

Objective To characterize a possible retention function of unique sequence in the 5'end of rat testis GABAA receptor β3t splicing variantMethods Rat testis GABAA receptor β3t splicing variant cDNA was cloned and two eukaryotic expression recombinant plasmids of pEGFP-N1 and pEGFP-C1 were constructed respectively by fusing green fluorescent protein to the N or C-terminus of β3t isoform. The recombinant plasmids were transfected into CHO cells by calcium phosphate co-precipitation method. Fluorescence microscope and laser confocal microscope were used to analyze localization of β3t in the transfected cells. ConA-Texas-Red was used to label cell ER and the localization of rat testis β3t splicing variant in CHO cells was determined.Results When rat testis β3t splicing variant was expressed in CHO cells, two expression patterns were delineated, the distributions of uniform and mainly discrete intracellular compartments respectively. The chimera product failed to be translocated into the cell surface when expressed in CHO cells; whereas the β3 subunit of rat brain was incorporated into the plasma membrane.Conclusion The inability of β3t to target into the ER may be a consequence of the unique 25 specific amino acid segments in the N terminus.     

Biochemical analysis of plasma-soluble invariant chains and their complex formation with soluble HLA-DR

The invariant chain (CD74) is preferentially localized in the cytoplasm and regulates the loading of exogenous derived peptides into HLA class II heterodimers. In addition, a small proportion of CD74:class II complexes is also expressed on the cell surface. We identified and quantified soluble CD74 (sCD74) molecules in the plasma and sCD74:sHLA-DR complexes by ELISA. EDTA plasma samples from 86 healthy probands were analyzed. sCD74 could be detected in all samples with a mean concentration of 1.14 relative units±1.04 SD (range 0.17-4.31). Approximately 10% of the samples had increased amounts of sCD74 (3.0 relative units). Complexes of sCD74 and sHLA-DR were detected in all samples and their quantities were positively correlated (r=0.83, p0.001) with the sCD74 concentrations. SDS-PAGE analysis of plasma samples with high sCD74 concentrations (3.0 relative units) revealed four isoforms of sCD74 with molecular weights of 45, 43, 35, 31 kDa corresponding to known sizes of intracellular CD74. However, only molecular weights of the 45 and 43 kDa isoforms of sCD74 are found complexed with sHLA-DR. Our data demonstrate, that CD74 molecules are present in their soluble form in the plasma of healthy probands and form complexes with soluble HLA-DR molecules.     

Self-splicing activity of the mitochondrial group-I introns from Aspergillus nidulans and related introns from other species

The mitochondrial genome of Aspergillus nidulans contains several group-I introns. Each one has been assayed for its ability to self-splice in vitro in the absence of proteins. The intron from the apocytochrome b gene is unusual among subgroup IB4 introns in being able to self-splice, unlike a similar intron from Saccharomyces cerevisiae. The first intron in the cytochrome oxidase subunit-1 gene self-splices but only correctly completes the first step of splicing; cryptic 3′ splice-sites are recognized instead and these are also used at a low frequency in vivo. The highly homologous intron from Podospora anserina completes both steps in vitro. The remaining introns do not self-splice. The correlation between subgroup category, the likely presence of specific tertiary interactions, and self-splicing activity is discussed. Received: 16 May / 25 August 1997     

Characterization of htAKR, a novel gene product in the aldo-keto reductase family specifically expressed in human testis

In human testis, expression of a novel member of the aldo-keto reductase family was identified. Based on its testis-specific expression, we termed this protein human testis aldo-keto reductase (htAKR). In addition to four major isoforms, the existence of multiple alternatively spliced products of htAKR was detected using RT-PCR followed by nested PCR. htAKR was a homologue of mouse liver keto-reductase, AKR1E1, with close similarity in their genomic organizations. htAKR4, the longest isoform, was expressed as a non-fused native form. It exhibited a limited activity toward 9,10-phenanthrenequinone, while no activity toward the steroids or prostaglandins was demonstrated. Using the laser capture microdissection technique and RT-PCR, expression of htAKR was detected in testicular germ cells as well as in interstitial cells. The levels of htAKR mRNA in the tissues obtained from seminoma were much lower than those in normal testes. A significant decline in the htAKR expression was observed when NEC8, a cell line originated from a human testicular germ cell tumour, was exposed to phorbol 12-myristate 13-acetate or 5alpha-dihydrotestosterone. These results indicate that the expression of htAKR, down-regulated in the testicular tumour, is possibly controlled by mitogenic and hormonal signals.