Exon junction complexes mediate the enhancing effect of splicing on mRNA expression

Intron-containing genes are generally expressed more effectively in human cells than are intronless versions of the same gene. We have asked whether this effect is due directly to splicing or instead reflects the action of components of the exon junction complex (EJC) that is assembled at splice junctions after splicing is completed. Here, we show that intron removal does not enhance gene expression if EJC formation is blocked. Conversely, RNA tethering of the EJC components SRm160 or RNPS1 boosts the expression of intronless mRNAs but not of spliced mRNAs. Splicing and RNPS1 tethering are shown to enhance the same steps in mRNA biogenesis and function, including mRNA 3' end processing and translation. Together, these data argue that the EJC is primarily responsible for the positive effect of splicing on gene expression.     

Exon shuffling mimicked in cell culture.

Undesired side products of DNA transfections are usually discarded. However, here, we show that such products may provide insight into mutational events that are also a major driving force in protein evolution. While studying the small heat-shock protein alphaA-crystallin, we transfected the hamster alphaA-crystallin gene into a mouse muscle cell line. One of the stable transfected cell lines expressed, in addition to the expected normal alphaA- and alternatively spliced alphaAins-crystallins, two slightly larger, immunologically cross-reacting proteins. These proteins were found to be encoded by a mutant alphaA-crystallin gene with a large intragenic duplication, arisen by illegitimate recombination at two CCCAT homologies, approximately 1.8 kilobases apart in the normal hamster alphaA-crystallin gene. As a consequence, a tandem-duplicated exon 3 sequence is present in the mature mRNA of this gene, resulting in a 41-residue repeat in the translated proteins. Cells expressing the elongated alphaA-crystallins have normal growth characteristics and the usual diffuse cytoplasmic distribution of immunoreactive alphaA-crystallin. Size-exclusion chromatography of cell extracts indicated that the mutant proteins are readily incorporated into the normal large water-soluble alphaA-crystallin complexes, showing that the insert does not disturb the integrity of these complexes. This viable alphaA-crystallin mutant thus mimics the origins and effects of exon duplication, which is a common consequence of exon shuffling in mammalian genome evolution.     

The repetition of large-earthquake ruptures

This survey of well-documented repeated fault rupture confirms that some faults have exhibited a "characteristic" behavior during repeated large earthquakes--that is, the magnitude, distribution, and style of slip on the fault has repeated during two or more consecutive events. In two cases faults exhibit slip functions that vary little from earthquake to earthquake. In one other well-documented case, however, fault lengths contrast markedly for two consecutive ruptures, but the amount of offset at individual sites was similar. Adjacent individual patches, 10 km or more in length, failed singly during one event and in tandem during the other. More complex cases of repetition may also represent the failure of several distinct patches. The faults of the 1992 Landers earthquake provide an instructive example of such complexity. Together, these examples suggest that large earthquakes commonly result from the failure of one or more patches, each characterized by a slip function that is roughly invariant through consecutive earthquake cycles. The persistence of these slip-patches through two or more large earthquakes indicates that some quasi-invariant physical property controls the pattern and magnitude of slip. These data seem incompatible with theoretical models that produce slip distributions that are highly variable in consecutive large events.     

Adults' word recall and word repetition

This experiment investigated age group differences in working memory by examining the effects of word length on adults' recall span and repetition rate. College students and adults, 60 to 94 years of age, recalled lists of one-, two-, or three-syllable words and repeated aloud pairs of one-, two- or three-syllable words. Word recall spans and word repetition rates were computed. Main effects of age group and word length were obtained on both measures, although the interactions were not significant. A second analysis examined the relationship between individuals' recall span and their repetition rate. Across all age groups combined, recall span was a linear function of repetition rate and accounted for 25 percent of the variance on the recall task. A reanalysis of the word repetitions revealed that older adults' word durations and inter-word pauses are longer than young adults'.     

Recovering sound sources from embedded repetition

Cocktail parties and other natural auditory environments present organisms with mixtures of sounds. Segregating individual sound sources is thought to require prior knowledge of source properties, yet these presumably cannot be learned unless the sources are segregated first. Here we show that the auditory system can bootstrap its way around this problem by identifying sound sources as repeating patterns embedded in the acoustic input. Due to the presence of competing sounds, source repetition is not explicit in the input to the ear, but it produces temporal regularities that listeners detect and use for segregation. We used a simple generative model to synthesize novel sounds with naturalistic properties. We found that such sounds could be segregated and identified if they occurred more than once across different mixtures, even when the same sounds were impossible to segregate in single mixtures. Sensitivity to the repetition of sound sources can permit their recovery in the absence of other segregation cues or prior knowledge of sounds, and could help solve the cocktail party problem.     

Stimulus repetition modulates gamma-band synchronization in primate visual cortex

When a sensory stimulus repeats, neuronal firing rate and functional MRI blood oxygen level-dependent responses typically decline, yet perception and behavioral performance either stay constant or improve. An additional aspect of neuronal activity is neuronal synchronization, which can enhance the impact of neurons onto their postsynaptic targets independent of neuronal firing rates. We show that stimulus repetition leads to profound changes of neuronal gamma-band (∼40–90 Hz) synchronization. Electrocorticographic recordings in two awake macaque monkeys demonstrated that repeated presentations of a visual grating stimulus resulted in a steady increase of visually induced gamma-band activity in area V1, gamma-band synchronization between areas V1 and V4, and gamma-band activity in area V4. Microelectrode recordings in area V4 of two additional monkeys under the same stimulation conditions allowed a direct comparison of firing rates and gamma-band synchronization strengths for multiunit activity (MUA), as well as for isolated single units, sorted into putative pyramidal cells and putative interneurons. MUA and putative interneurons showed repetition-related decreases in firing rate, yet increases in gamma-band synchronization. Putative pyramidal cells showed no repetition-related firing rate change, but a decrease in gamma-band synchronization for weakly stimulus-driven units and constant gamma-band synchronization for strongly driven units. We propose that the repetition-related changes in gamma-band synchronization maintain the interareal stimulus signaling and sharpen the stimulus representation by gamma-synchronized pyramidal cell spikes.Stimulus repetition typically leads to reduced neuronal firing rates and reduced functional MRI blood oxygen level-dependent signals, whereas behavior that is based on stimulus processing is not affected or is enhanced (1). Different models have been proposed to reconcile these behavioral and neurophysiological findings (1). In a “fatigue model,” neuronal responses are reduced in proportion to their amplitude, leaving relative response patterns unchanged; in a “sharpening model,” neurons that code features irrelevant to identification of a stimulus exhibit repetition suppression, leading to a sparser and sharpened representation of the repeated stimulus; and in a “facilitation model,” stimulus repetition leads to faster stimulus processing, and thereby smaller overall neuronal activity. Gotts and coworkers (2–4) suggested a “synchronization model” in which stimulus repetition leads to reduced firing rates accompanied by increased synchronization. The increased synchronization might explain how less-activated neuronal groups can maintain their impact onto postsynaptic neurons and, ultimately, behavior, while reducing metabolic costs at the same time. The synchronization model has received support from a number of studies in human subjects, using source-localized magnetoencephalography. Ghuman et al. (5) report enhanced frontotemporal 14-Hz synchronization for repeated vs. novel stimuli. Gilbert et al. (3) found that stimulus repetition leads to enhanced 5- to 15-Hz power in the right fusiform gyrus and enhanced 15- to 35-Hz power in striate and extrastriate cortex. Corresponding data were also reported for multisite microelectrodes recordings in striate and parietal cortex of awake cats, where von Stein et al. (6) found that interareal alpha-band synchronization was stronger for repeated compared with novel stimuli. The common finding across these studies is enhanced alpha/beta activity or coupling for repeated stimuli. The alpha coupling reported by von Stein et al. (6) occurs in a behavioral context and has a phase relationship and layer specificity that suggests a top-down–directed interaction. Thus, enhanced alpha/beta activity or coupling for repeated stimuli might reflect enhanced top-down signaling, perhaps related to enhanced predictability of repeated stimuli. However, increased synchronization with stimulus repetition according to the model of Gotts and coworkers (2–4) should also serve the maintenance of feedforward signaling of repeated stimuli in the face of reduced firing rates. Feedforward signaling has been strongly linked to local and interareal gamma-band synchronization (7–9). Local gamma-band synchronization likely enhances the postsynaptic impact of the precisely synchronized output spikes (10). Interareal gamma-band synchronization likely aligns excitability cycles such that inputs arrive when postsynaptic target neurons are receptive (11, 12). However, whether multiple presentations of a stimulus result in enhanced gamma-band synchronization remains unknown (details are provided in SI Discussion). We investigated gamma-band synchronization within and between macaque monkey areas V1 and V4 and report that stimulus repetition leads to profound changes in gamma-band synchronization within and between these areas.     

外显子测序技术在一个尿道下裂家系基因分析及产前诊断中的应用

目的采用外显子测序(exon sequencing,ES)联合Sanger测序技术检测1个尿道下裂家系的致病基因,并对这一致病基因进行产前诊断。方法采集先证者及其父母的外周血,并提取DNA。对先证者的DNA进行性腺疾病相关基因的外显子测序找到致病基因,并针对该致病基因对先证者父母的DNA进行Sanger验证,明确是遗传自亲代。对先证者母亲再次妊娠的羊水标本提取DNA,采用Sanger测序对该致病基因行产前诊断。结果先证者SRD5A2(NM_000348):c.607GA,P.(Gly203Ser)杂合突变及c.680GA,P.(Arg227Gln)杂合突变组成的复合杂合突变。而其表型正常的母亲为SRD5A2(NM_000348):c.607GA,P.(Gly203Ser)杂合突变;其表型正常的父亲为SRD5A2(NM_000348):c.680GA,P.(Arg227Gln)杂合突变。先证者母亲再次妊娠产前诊断结果为SRD5A2(NM_000348):c.680GA,P.(Arg227Gln)杂合突变。结论 ES联合Sanger测序可用于检测尿道下裂家系的致病基因及进行产前诊断。且此方法快速、准确、经济。     

非小细胞肺癌EGFR 20号外显子插入突变靶向治疗的研究进展

摘 要：表皮生长因子受体20号外显子插入突变（epidermal growth factor receptor exon 20 insertions,EGFR ex20ins）是非小细胞肺癌中的一种罕见突变。由于EGFR ex20ins对EGFR-酪氨酸激酶抑制剂、化疗以及免疫治疗普遍不敏感,因此一直是临床治疗面临的难题。近年来,EGFR ex20ins靶向治疗研究进展不断,为该类患者的治疗带来新的希望。全文对EGFR ex20ins靶向治疗进展和临床研究进行综述,为临床靶向治疗提供参考。     

Exon shuffling generates an immunoglobulin heavy chain gene.

From endonuclease EcoRI partial libraries of DNAs from mouse embryo and MOPC 141, a gamma 2b-producing myeloma, clones were isolated by using a DNA fragment carrying the gamma 2b constant (C) region gene as a hybridization probe. One clone from MOPC 141 contained a heavy chain variable (V) gene and the C gamma 2b gene, as demonstrated by R-loop mapping. The V gene and C gene in this clone were separated by a 3.9-kilobase intron. The characterization of this clone as well as the embryonic clones suggest that at least two recombination events occurred to create the gamma 2b gene in MOPC 141. One of the events is analogous to the V-J joining previously demonstrated in the light chain genes, which brings the major part of the V gene next to a short coding sequence (J). The other event we refer to as "C mu-C gamma 2b switch recombination" because a portion of the intron between the V gene and C gene of the rearranged gamma 2b gene is derived from the 5' flanking sequence of the embryonic C mu gene. A model suggesting how the phenomenon of switch seen in lymphocytes may occur is presented.     

Exon 6 and 2 peroxisome proliferator-activated receptor-gamma polymorphisms in polycystic ovary syndrome

Obesity affects about 44% of women with polycystic ovary syndrome (PCOS). Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is one of the genes involved in the differentiation of adipose tissue. In an attempt to shed light on the high percentage of obesity in PCOS, we examined polymorphisms at exons 6 and 2 of the PPAR-gamma gene in 100 PCOS patients and in 100 healthy controls matched for age and body mass index (BMI). The T allele frequency of exon 6 was significantly higher (P < 0.05) in PCOS patients compared with control women. In addition, the BMI and leptin levels were significantly higher (P < 0.05) in PCOS patients carrying the C-->T substitution than in controls. There was no significant difference in leptin levels after normalization for BMI. The Pro(12)Ala polymorphism at exon 2 was unrelated to BMI and/or leptin levels in PCOS women. In conclusion, the higher frequency of the C-->T substitution in exon 6 of the PPAR-gamma gene in PCOS women suggests that it plays a role in the complex pathogenetic mechanism of obesity in PCOS, whereas the Pro(12)Ala polymorphism does not seem to affect BMI in PCOS women.     

Visualization of an inverted terminal repetition in vaccinia virus DNA.

An inverted terminal repetition was observed in DNA molecules extracted from vaccinia virus. The repeated sequence was visualized by (i) nicking the hairpin loops present of the ends of vaccinia virus DNA, (ii) separating the strands of DNA by alkali denaturation, (iii) allowing the single strands to self-anneal, and (iv) examining the DNA with an electron microscope. Single-stranded circular molecules, each of which contained a duplex projection (3.54 +/- 0.12 micron) representing the terminal repetition, readily formed. Similar size projections were also seen in heteroduplex structures formed by crosshybridization of the separated strands of the two terminal HindIII restriction fragments. Based on contour length measurements and the electrophoretic mobility of the isolated inverted terminal repetition, a molecular weight of approximately 6.9 X 10(6), equivalent to about 10,500 nucleotide base pairs, was estimated. Evidence was obtained from DNA-RNA hybridization studies that the terminal repetition is transcribed.     

Exon skipping by overexpression of a Drosophila heterogeneous nuclear ribonucleoprotein in vivo.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are abundant RNA-binding proteins that are implicated in splicing regulation. Here we investigate the role of a Drosophila hnRNP in splicing regulation in living animals. We find that overexpression of the Drosophila hnRNP HRB98DE leads to skipping of all internal exons in the Drosophila dopa decarboxylase (Ddc) pre-mRNA in vivo. These results indicate that HRB98DE has a splicing activity that promotes use of terminal splice sites. The effect of excess HRB98DE on Ddc splicing is transient, even though high levels of HRB98DE persist for at least 24 hr. This suggests that Drosophila larvae can induce a compensating mechanism to counteract the effects of excess HRB98DE.     

MEF2A基因第11号外显子突变的检测及其基因结构分析

目的检测我国汉族人群中MEF2A基因第11号外显子区的突变,分析MEF2A特异性突变的基因结构和遗传学意义。方法用PER-SSCP和／或PCR产物直接测序法对536例冠状动脉粥样硬化性心脏病阳性病例、232例冠状动脉粥样硬化性心脏病阴性对照及232例健康体检者的MEF2A基因第11号外显子区进行突变检测,用质粒克隆测序法对各突变位点作进一步验证。结果在冠状动脉粥样硬化性心脏病阳性病例组发现一例MEF2A 21碱基的特异性突变。另外还发现二种罕见的突变类型。结论MEF2A基因第11号外显子区存在多种罕见的突变类型,基因结构分析显示MEF2A基因21碱基特异性突变有多种形成模式。     

Origins of metabolic diversity: Evolutionary divergence by sequence repetition

Recurring patterns of primary structure have been observed in enzymes that mediate sequential metabolic reactions in bacteria. The enzymes, muconolactone Delta-isomerase [(+)-4-hydroxy-4-carboxymethylisocrotonolactone Delta(2)-Delta(3)-isomerase, EC 5.3.3.4] and beta-ketoadipate enol-lactone hydrolase [4-carboxymethylbut-3-enolide(1,4)enol-lactone-hydrolase, EC 3.1.1.24], have been coselected in bacterial populations because the isomerase can confer no nutritional advantage in the absence of the hydrolase. Similar amino acid sequences recur within the structure of the isomerase, and the amino-terminal amino acid sequence of the isomerase from Pseudomonas putida appears to be evolutionarily homologous with the corresponding sequence of a beta-ketoadipate enol-lactone hydrolase from Acinetobacter calcoaceticus. One interpretation of the sequence repetitions is that they reflect tandem duplication mutations that took place early in the evolution of the proteins. According to this view, the mutations caused elongation of structural genes and the creation of duplicated genes as the metabolic pathways evolved. A review of the sequence data calls attention to a different hypothesis: repeated amino acid sequences were introduced in the course of the proteins' evolution by substitution of copies of DNA sequences into structural genes. Our observations are interpreted on the basis of a model proposing genetic exchange between misaligned DNA sequences. The model predicts that misalignments in one chromosomal region can influence the nature of mutations in another region. Thus, as often has been observed, the mutability of a base pair will be determined by its location in a DNA sequence. Furthermore, the intrachromosomal recombination of DNA sequences may account for complex genetic modifications that occur as new pathways evolve. The model provides an interpretation of an apparent paradox, the rapid creation of new metabolic traits by bacterial genomes that are remarkably resistant to genetic drift.