激光对家蚕诱变效应的探讨

激光应用于生物学的研究,国内外都取得了一定进展。Zkzolo用红宝石激光器照射雄性果蝇幼虫产生了突变。中山大学用CO_2激光器处理植物花药,引起大量染色体畸变。安徽农学院用钕玻璃激光器照射蚕卵,获得一雌一雄的突变体,进而育成新原种。华南农学     

激光在蚕业上的应用试验

为了探索激光在蚕业上的应用效应,我们从1974年开始,在中国科学院吉林应用化学研究所、吉林农业大学等单位大力协作下,先后用多种激光器在柞蚕、桑蚕上进行增产增丝和诱变育种实验。五年来,通过小区实验和农村基点小量试验鉴定,初步证明了应用一定剂量的钕玻璃激光辐照蚕卵,具有提高产茧量、改进丝质和诱发突变作用;应用氮分子激光辐照柞蚕蛹和桑蚕卵也诱发了突变个体,并从这些突变体中初步选育出新类型。     

ABCA1胞外第四环缺失突变体的构建及其抗砷性初步研究

目的：构建ABCA1细胞外第四环第1 479～1 597位氨基酸残基缺失的突变体。方法：采用重叠区扩增基因拼接法构建ABCA1第1 479～1 597位氨基酸残基缺失的突变体基因,并将其克隆至pcDNA3.1/V5-His/ABCA1重组载体上,脂质体法转染Hela细胞,激光共聚焦观察突变体定位,Cell Counting Kit-8(CCK-8)试剂盒检测其48h急性砷中毒后生存率的变化。结果：该突变体基因经DNA序列分析表明其具有正确的序列和阅读框。激光共聚焦证实其表达的蛋白仍然能正确定位在Hela细胞的细胞膜上。CCK-8结果显示转染重组质粒和突变体质粒的细胞在各种砷浓度下生存率都较空载体组高。结论：成功构建了ABCA1胞外第四环第1 479～1 597位氨基酸残基缺失的突变体,且其表达的蛋白仍然定位于细胞膜上,突变体仍然具有一定的抗砷性,提示ABCA1胞外第四环可能不是关键抗砷结构域。     

激光诱变家蚕突变及其遗传育种的研究

本文报道了激光诱导家蚕单性生殖五年（１９８９—１９９３）来的研究结果。由于家蚕雌雄核受刺激的敏感性存在着差异,激光刺激不仅能使未受精的处女蛾卵发育产生孤雌生殖;而且能够破坏交配蛾卵的雌核发育产生孤雄生殖。试验结果说明激光诱发家蚕单性生殖具有明显效果,现已选育成“单性１号”和“单性２号”两个新突变体及其优良杂交组合“单性１号×皖５”和“单性１号×中８”,由于“单性１号”是斑纹限性品种,用它作母本的杂交种可为蚕业生产带来很大的经济效益。     

YlRas2调控解脂耶氏酵母二型性转换的下游作用分子—Hoy1的鉴定

[目的]寻找解脂耶氏酵母中受YlRas2控制的参与菌丝形成调控的新基因,揭示新的受YlRas2控制的信号传递途径。[方法]在菌丝形成能力缺陷的Ylras2"突变体中进行基因的随机过量表达,筛选菌丝形成能力获得回复的突变体,通过TAIL-PCR方法鉴定突变体中发生过量表达的基因。[结果]筛选得到27个菌丝形成能力获得回复的突变体,经鉴定其中的一个突变体发生过量表达的基因为HOY1,它编码一个转录因子,经确认HOY1基因的过量表达的确可以回复Ylras2"突变体的菌丝形成缺陷。HOY1基因的启动子序列分析和转录活性检测结果都显示HOY1可能是转录因子Mhy1调控的靶基因。[结论]Hoy1和Mhy1可能在YlRas2的下游发挥作用,它们可能是YlRas2调控菌丝形成的新信号传递途径的成员。     

植物体内的叶绿素降解与滞绿突变体

文章介绍植物体内叶绿素降解途径和滞绿突变体的新类型研究进展。     

辐射诱发唐菖蒲复色花突变体的AFLP分析

以唐菖蒲品种'新秀'的种球为材料,经不同剂量50Co γ射线诱变后,进行生物学性状观察,并以'新秀'为对照.用AFLP分子标记对突变体进行多态性分析.结果显示,(1)诱变后代出现了广泛变异,并从75 GY剂量组M:代中选出了花瓣呈粉白相间的复色花突变体.(2)突变体的大多数引物扩增产物带型与对照带型差异显著;64种引物中50对引物检测出DNA分子的多态性,产生1 600条清晰谱带,多态性位点112个,多态性13.08%,相似性系数为93%.差异片段主要集中于100～700 bp之间.研究表明,复色花突变体和对照之间的差异与遗传物质的改变相关,其中一些可能与花色形成基因有关.     

铜绿假单胞菌多重耐药基因的筛选及鉴定

[目的]研究铜绿假单胞菌中与耐药性相关的基因.[方法]筛选转座突变体文库中对多种抗菌药物敏感的突变体,通过随机PCR、核苷酸测序及序列比对确定突变体中转座子的插入位点及其破坏的基因.[结果]筛选得到2株对多种抗菌药物敏感的突变体,其中被破坏的基因分别为功能未知的新基因PA2580和PA2800.[结论]PA2580和PA2800可能分别通过参与细胞氧化还原作用和细胞壁合成进而与铜绿假单胞菌耐药性相关.     

空间诱变水稻大粒型突变体的遗传育种研究

对粳稻农垦58经空间诱变产生的大粒型突变体进行大粒型性状的遗传分析和育种应用研究。结果表明,大籽型突变体的籽粒大小（以籽粒体积表示）表现为受多基因控制的数量性状。大粒型突变体谷粒细长,具馨香味,外观品质优,对粳稻的亲和性好,是一个罕见的优质米资源。以大粒型突变体为供体,高产、外观米质差的晚粳推广品种为受体,采用不饱和回交结合分离世代糙米外观品质鉴定的方法,将大粒型突变体的优质性状导入晚粳背景,选育出外观米质优的晚粳新品系。对采用不饱和回方法改良数量性状进行了讨论。     

激光对家蚕诱变效应的探讨

激光应用于生物学的研究,国内外都取得 了一定进展。Zkzolot^[8]用红宝石激光器照射雄 性果蝇幼虫产生了突变。中山大学^[3]用CO₂激 光器处理植物花药,引起大量染色体畸变。安 徽农学院^[5]用钦玻璃激光器照射蚕卵,获得一 雌一雄的突变体,进而育成新原种。华南农学 院^[2]用显离子激光照射蓖麻蚕蛹,诱发蛾翅突 变,并育成优良新品种。但也有经照射后未发 现突变的>,对此有必要从实践和理论上作进 一步探讨。我们采用特定位点法,以家蚕形质 性状为标志,研究了激光对家蚕诱变的效应,并 进行了染色体观察,现将结果报告如下.     

激光诱发家蚕孤雌生殖的研究：Ⅲ单性一号及单性二号性状表现

采用CO_2激光扩束辐照诱发家蚕孤雌生殖,从获得的单性蚕中,选择出单性一号和单性二号两个类型,单性一号由血色限性突变为斑纹限性,体质强健好养,单性二号仍保持亲本的血色限性,但茧丝质有了提高。     

The pilus-induced Ca2+ flux triggers lysosome exocytosis and increases the amount of Lamp1 accessible to Neisseria IgA1 protease

The IgA1 protease secreted by the pathogenic Neisseriae cleaves Lamp1, a major integral membrane glycoprotein of lysosomes, and significantly reduces its steady-state levels in an infected cell. IgA1 protease hydrolysis of Lamp1 is inefficient at the low pH of lysosomes, strongly suggesting that the enzyme is unlikely to reduce Lamp1 levels within lysosomes to any appreciable extent. We therefore explored the possibility that the protease may reach Lamp1 through an alternative route. We demonstrate that Neisseria pili induce a transient increase in the levels of cytosolic free Ca²⁺ in A431 human epithelial cells, as demonstrated previously for ME180 cells. This Ca²⁺ flux triggers lysosome exocytosis, quickly altering the cellular distribution of Lamp1 and increasing surface Lamp1 levels. Finally, we demonstrate that surface Lamp1 is cleaved by IgA1 protease secreted by adherent bacteria. We conclude that the pilus-induced Ca²⁺ flux increases the amount of Lamp1 that is cleavable by the IgA1 protease.     

Lysosomal membranes from beige mice contain higher than normal levels of endoplasmic reticulum proteins

Chediak-Higashi syndrome is characterized by dysfunctional giant organelles of common origin, that is, lysosomes, melanosomes, and platelet dense bodies. Its defective gene LYST encodes a large molecular weight protein whose function is unknown. The Beige mouse also defective in Lyst is a good model of the human disease. Purified lysosomes from Beige and normal black mouse livers were used to carry out a proteomics study. Two-dimensional gel electrophoretic separation of soluble lysosomal proteins of Beige and normal mice revealed no major differences. The cleavable isotope-coded affinity tag (cICAT) technique was used to compare the composition of Beige and normal lysosomal membrane proteins. While the levels of common proteins, that is, Lamp1, Lamp2, and Niemann-Pick type C1, were decreased in Beige mice, there was an increase in the levels of endoplasmic reticulum (ER) resident proteins, for example, cytochrome P450, NADPH-cytochrome P450 oxidoreductase, and flavin-containing monooxygenase. Confocal microscopy confirmed that another ER protein, calnexin, colocalizes with Lamp1 on membranes of giant lysosomes from fibroblasts of Chediak-Higashi syndrome patient. Our results suggest that LYST may play a role in either preventing inappropriate incorporation of proteins into the lysosomal membrane or in membrane recycling/maturation.     

A determinant in the cytoplasmic tail of the cation-dependent mannose 6- phosphate receptor prevents trafficking to lysosomes

The bovine cation-dependent mannose 6-phosphate receptor (CD-MPR) is a type 1 transmembrane protein that cycles between the trans-Golgi network, endosomes, and the plasma membrane. When the terminal 40 residues were deleted from the 67-amino acid cytoplasmic tail of the CD- MPR, the half-life of the receptor was drastically decreased and the mutant receptor was recovered in lysosomes. Analysis of additional cytoplasmic tail truncation mutants and alanine-scanning mutants implicated amino acids 34-39 as being critical for avoidance of lysosomal degradation. The cytoplasmic tail of the CD-MPR was partially effective in preventing the lysosomal membrane protein Lamp1 from entering lysosomes. Complete exclusion required both the CD-MPR cytoplasmic tail and transmembrane domain. The transmembrane domain alone had just a minor effect on the distribution of Lamp1. These findings indicate that the cytoplasmic tail of the CD-MPR contains a signal that prevents the receptor from trafficking to lysosomes. The transmembrane domain of the CD-MPR also contributes to this function.     

Follicular atresia in the prepubertal spiny mouse (<Emphasis Type="Italic">Acomys cahirinus</Emphasis>) ovary

This study was designed to determine follicular atresia in the newborn and the prepubertal spiny mouse. We analyzed the processes of follicle loss using classical markers of apoptosis (TUNEL reaction, active caspase-3) and autophagy (Lamp1). Numerous small clear vacuoles and autophagosomes as well as strong Lamp1 staining were observed in dying oocytes of all follicle types, especially of the primordial and primary ones. Active caspase 3 and the TUNEL reaction were detected only in the granulosa cells of large secondary and antral follicles. The expression of apoptosis and autophagy markers was also changing during the prepubertal period. Western blot analysis indicated that at the moment of birth, females undergo an increased rate of follicular atresia mediated by autophagy, while apoptosis is the dominant form of ovarian atresia in consecutive postnatal days. On the basis of these observations, we concluded that apoptosis and autophagy are involved in follicular atresia and these processes are cell and developmental stage-specific.     

Lysosome associated membrane protein 1 (Lamp1) traffics directly from the TGN to early endosomes

The precise trafficking routes followed by newly synthesized lysosomal membrane proteins after exit from the Golgi are unclear. To study these events we created a novel chimera (YAL) having a lumenal domain comprising two tyrosine sulfation motifs fused to avidin, and the transmembrane and cytoplasmic domains of lysosome associated membrane protein 1 (Lamp1). The newly synthesized protein rapidly transited from the trans- Golgi Network (TGN) to lysosomes (t(1/2) approximately 30 min after a lag of 15-20 min). However, labeled chimera was captured by biotinylated probes endocytosed for only 5 min, indicating that the initial site of entry into the endocytic pathway was early endosomes. Capture required export of YAL from the TGN, and endocytosis of the biotinylated reagent, and was essentially quantitative within 2 h of chase, suggesting that all molecules were following an identical route. There was no evidence of YAL trafficking via the cell surface. Fusion of TGN-derived vesicles with 5 min endosomes could be recapitulated in vitro, but neither late endosomes nor lysosomes could serve as acceptor compartments. This suggests that contrary to previous conclusions, most if not all newly synthesized Lamp1 traffics from the TGN to early endosomes prior to delivery to late endosomes and lysosomes.     

Autophagy maturation associated with CD38‐mediated regulation of lysosome function in mouse glomerular podocytes

Podocytes are highly differentiated glomerular epithelial cells that contribute to the glomerular barrier function of kidney. A role for autophagy has been proposed in maintenance of their cellular integrity, but the mechanisms controlling autophagy in podocytes are not clear. The present study tested whether CD38‐mediated regulation of lysosome function contributes to autophagic flux or autophagy maturation in podocytes. Podocytes were found to exhibit a high constitutive level of LC3‐II, a robust marker of autophagosomes (APs), suggesting a high basal level of autophagic activity. Treatment with the mTOR inhibitor, rapamycin, increased LC3‐II and the content of both APs detected by Cyto‐ID Green staining and autophagolysosomes (APLs) measured by acridine orange staining and colocalization of LC3 and Lamp1. Lysosome function inhibitor bafilomycin A1 increased APs, but decreased APLs content under both basal and rapamycin‐induced conditions. Inhibition of CD38 activity by nicotinamide or silencing of CD38 gene produced the similar effects to that bafilomycin A1 did in podocytes. To explore the possibility that CD38 may control podocyte autophagy through its regulation of lysosome function, the fusion of APs with lysosomes in living podocytes was observed by co‐transfection of GFP‐LC3B and RFP‐Lamp1 expression vectors. A colocalization of GFP‐LC3B and RFP‐Lamp1 upon stimulation of rapamycin became obvious in transfected podocytes, which could be substantially blocked by nicotinamide, CD38 shRNA, and bafilomycin. Moreover, blockade of the CD38‐mediated regulation by PPADS completely abolished rapamycin‐induced fusion of APs with lysosomes. These results indicate that CD38 importantly control lysosomal function and influence autophagy at the maturation step in podocytes.     

Rhophilin-2 is targeted to late-endosomal structures of the vesicular machinery in the presence of activated RhoB

Rhophilin-2 or p76(RBE), a protein whose expression is induced by the cyclic AMP pathway in thyrocytes, contains several protein-protein interaction domains including HR-1, Bro1 and PDZ domains, and is a partner of RhoB in its GTP-bound form (Eur J Biochem, 269(24): 6241-9, 2002). We here define its subcellular localization and dissect the significance of its domains. By subcellular fractionation and colocalization experiments, rhophilin-2 is recruited to subcellular organelles by activated RhoB-GTP. As for its yeast homologue, Npi3/Bro1p, the Bro1 domain of rhophilin-2 is necessary to its recruitment to the vesicular structures, which are not labeled for EEA1 nor Lamp1, but well with the late endosome marker CD63.     

Unusual intracellular trafficking of Salmonella typhimurium in human melanoma cells

Salmonella spp. are enterobacteria capable of invading and replicating in both professional and non‐professional phagocytes. Here, we investigate the fate of S. typhimurium in human melanoma MelJuSo cells. The bacterium entered MelJuSo cells by a trigger mechanism and resided within a unique organelle, the Salmonella‐containing vacuole (SCV). The SCV acquired early endosomal markers transiently and then underwent a series of membrane modifications. In HeLa cells, vacuole maturation is characterized by the simultaneous acquisition of the lysosomal membrane glycoproteins (Lgps) Lamp1, CD63 and vacuolar (v)‐ATPase; in MelJuSo cells, however, acquisition of CD63 and v‐ATPase preceded that of Lamp1. A very striking event in MelJuSo cells was the arrest of bacterial septation starting from 8 h after infection. Bacteria nevertheless continued to elongate, remained morphologically intact and viable and were eventually exocytosed. This original feature was observed in several skin‐related cells including melanocytes, suggesting that it may provide the basis for an efficient host defence mechanism against Salmonella infection.