剂量补偿和MSL复合物研究进展

剂量补偿效应(Dosage compensation effect)广泛存在于两性真核生物,是基于性别决定、平衡不同性别间基因转录水平的遗传效应。MSL复合物(Male-specific lethal complex)是果蝇剂量补偿机制的核心,它乙酰化雄性果蝇X染色体上一些特定的位点,双倍激活X连锁活跃基因的转录,从而弥补雄性果蝇只具有单一条X染色体的不足。目前,已对果蝇MSL复合物各主要成分进行了结构分析,大体了解了各组分间的相互作用位点,并对该复合物的识别机制进行了大量的研究。与果蝇不同,哺乳动物是通过雌性个体一条X染色体的失活来实现剂量补偿。虽然哺乳动物MSL复合物的组成已被鉴定,但对其功能的研究还处于初步阶段。迄今为止,对硬骨鱼类剂量补偿及MSL复合物的研究极少。文章概括了线虫、果蝇和哺乳动物各物种剂量补偿机制的异同,综述了果蝇MSL复合物及其剂量补偿机制作用机理的研究进展,并提出有待解决的问题,同时利用同线性分析发现了不同鱼类msl3基因的多样性,为今后继续研究各物种的剂量补偿机制提供基础资料和研究方向。     

Vaccinia virus G9 protein is an essential component of the poxvirus entry-fusion complex

The vaccinia virus G9R gene (VACWR087) encodes a protein of 340 amino acids with the following structural features that are conserved in all poxviruses: a site for N-terminal myristoylation, 14 cysteines, and a C-terminal transmembrane domain. Previous studies showed that G9 is one of eight proteins associated in a putative entry-fusion complex. Our attempt to isolate a mutant without the G9R gene was unsuccessful, suggesting that it is essential for virus replication. To further investigate its role, we constructed a recombinant vaccinia virus in which G9R is regulated by addition of an inducer. Induced G9 protein was associated with mature infectious virions and could be labeled with a membrane-impermeant biotinylation reagent, indicating surface exposure. Omission of inducer reduced the infectious-virus yield by about 1.5 logs; nevertheless, all stages of virus morphogenesis appeared normal and extracellular virions were present on the cell surface. Purified virions assembled without inducer had a specific infectivity of less than 5% of the normal level and a comparably small amount of G9, whereas their overall polypeptide composition, including other components of the entry-fusion complex, was similar to that of virions made in the presence of inducer or of wild-type virions. G9-deficient virions bound to cells, but penetration of cores into the cytoplasm and early viral RNA synthesis were barely detected, and cell-cell fusion was not triggered by low pH. Of the identified components of the multiprotein complex, G9 is the sixth that has been shown to be required for entry and membrane fusion.     

The NTPase/helicase activities of Drosophila maleless, an essential factor in dosage compensation.

Drosophila maleless (mle) is required for X chromosome dosage compensation and is essential for male viability. Maleless protein (MLE) is highly homologous to human RNA helicase A and the bovine counterpart of RNA helicase A, nuclear helicase II. In this report, we demonstrate that MLE protein, overexpressed and purified from Sf9 cells infected with recombinant baculovirus, possesses RNA/DNA helicase, adenosine triphosphatase (ATPase) and single-stranded (ss) RNA/ssDNA binding activities, properties identical to RNA helicase A. Using site-directed mutagenesis, we created a mutant of MLE (mle-GET) that contains a glutamic acid in place of lysine in the conserved ATP binding site A. In vitro biochemical analysis showed that this mutation abolished both NTPase and helicase activities of MLE but affected the ability of MLE to bind to ssRNA, ssDNA and guanosine triphosphate (GTP) less severely. In vivo, mle-GET protein could still localize to the male X chromosome coincidentally with the male-specific lethal-1 protein, MSL-1, but failed to complement mle1 mutant males. These results indicate that the NTPase/helicase activities are essential functions of MLE for dosage compensation, perhaps utilized for chromatin remodeling of X-linked genes.     

Characterization of an eppin protein complex from human semen and spermatozoa

Eppin (SPINLW1; serine peptidase inhibitor-like with Kunitz and WAP domains 1 (eppin); epididymal protease inhibitor) coats the surface of human ejaculate spermatozoa and originates from Sertoli and epididymal epithelial cells. In this study, we have isolated native eppin from ejaculate supernatants (seminal plasma) and washed ejaculate spermatozoa using column chromatography and two-dimensional SDS-PAGE, and identified by mass spectrometry and Western blots an eppin protein complex (EPC) containing lactotransferrin (LTF; also known as lactoferrin), clusterin (CLU), and semenogelin (SEMG1). To confirm the association of eppin with LTF, CLU, and SEMG1, antibodies to CLU and LTF were used to immunoprecipitate CLU and LTF from human sperm lysates. In both cases identical results were obtained, namely, the immunoprecipitate of the EPC. Additionally, we localized eppin, LTF, and CLU in human Sertoli cells and on human testicular and ejaculate spermatozoa, implying that the EPC is present on spermatozoa from the time they leave the seminiferous tubule. On ejaculate spermatozoa eppin, LTF, and CLU colocalize on the tail. The identification of the EPC components suggests that LTF, CLU, and/or eppin receptors may function as sperm plasma membrane receptors for the EPC, implicating the complex as a central player in a network of protein-protein interactions on the human sperm surface. The EPC may provide a surface network with microbicidal properties that protects spermatozoa as well as regulates the sperm's transition to a motile, capacitated sperm.     

''Sheltered disruption'' of Neurospora crassa MOM22, an essential component of the mitochondrial protein import complex.

MOM22 is a component of the protein import complex of the mitochondrial outer membrane of Neurospora crassa. Using the newly developed procedure of 'sheltered disruption', we created a heterokaryotic strain harboring two nuclei, one with a null allele of the mom-22 gene and the other with a wild-type allele. Homokaryons bearing the mom-22 disruption could not be isolated, suggesting that mom-22 is an essential gene. The mutant nucleus can be forced to predominate in the heterokaryon through the use of specific nutritional and inhibitor resistance markers. Cultivation of the heterokaryon under conditions favoring the mutant nucleus resulted in selective depletion of MOM22. MOM22-depleted cells did not grow and contained mitochondria with an altered morphology and protein composition. Protein import into isolated, MOM22-depleted mitochondria was abolished for most precursor proteins destined for all subcompartments. In contrast, precursors of MOM19, MOM22 and MOM72 became inserted normally into the outer membrane, defining a novel MOM22-independent import pathway which remained intact in mutant mitochondria. Furthermore, the specific binding of the ADP/ATP carrier to the outer membrane was unaffected, but subsequent transport across the outer membrane did not occur. Our data show that MOM22 is an essential component of Neurospora cells specifically required for the biogenesis of mitochondria.     

Vaccinia virus H2 protein is an essential component of a complex involved in virus entry and cell-cell fusion

The vaccinia virus H2R gene (VACWR 100) is conserved in all sequenced members of the poxvirus family and encodes a protein with a predicted transmembrane domain and four invariant cysteines. A recombinant vaccinia virus, in which expression of the H2 protein is stringently regulated, was unable to replicate without inducer. However, under nonpermissive conditions, all stages of virus morphogenesis appeared normal and extracellular virions were detected at the tips of actin tails. Nevertheless, virus did not spread to neighboring cells nor did syncytia form after low-pH treatment. Purified -H2 and +H2 virions from cells infected in the absence or presence of inducer, respectively, were indistinguishable in microscopic appearance and contained the same complement of major proteins, though only +H2 virions were infectious. The -H2 virions bound to cells, but their cores did not penetrate into the cytoplasm. In addition, exogenously added -H2 virions were unable to mediate the formation of syncytia after low-pH treatment. In contrast, virions lacking the A27 (p14) protein, which was previously considered to have an essential role in fusion, penetrated cells and induced extensive syncytia. The properties of H2, however, are very similar to those recently reported for the A28 protein. Moreover, coimmunoprecipitation experiments indicated an interaction between H2 and A28. Therefore, H2 and A28 are the only proteins presently known to be specifically required for vaccinia virus entry and are likely components of a fusion complex.     

Indian hedgehog is an essential component of mechanotransduction complex to stimulate chondrocyte proliferation

Indian hedgehog (Ihh), a member of the vertebrate hedgehog morphogen family, is a key signaling molecule that controls chondrocyte proliferation and differentiation. In this study, we show a novel function of Ihh. Namely, it acts as an essential mediator of mechanotransduction in cartilage. Cyclic mechanical stress greatly induces the expression of Ihh by chondrocytes. This induction is abolished by gadolinium, an inhibitor of stretch-activated channels. This suggests that the IHH gene is mechanoresponsive. The mechano-induction of Ihh is essential for stimulating chondrocyte proliferation by mechanical loading. The presence of an Ihh functional blocking antibody during loading completely abolishes the stimulatory effect of mechanical load on proliferation. Furthermore, Ihh mediates the mechanotransduction process in a bone morphogenic protein (BMP)-dependent and parathyroid hormone-related peptide-independent manner. BMP 2/4 are up-regulated by mechanical stress through the induction of Ihh, and BMP antagonist noggin inhibits mechanical stimulation of chondrocyte proliferation. This suggests BMP lies downstream of Ihh in mechanotransduction pathway. Our data suggest that Ihh may transduce mechanical signals during cartilage growth and repair processes.     

BLAP75, an essential component of Bloom's syndrome protein complexes that maintain genome integrity

Bloom's syndrome (BS) is a rare human genetic disorder characterized by dwarfism, immunodeficiency, genomic instability and cancer predisposition. We have previously purified three complexes containing BLM, the helicase mutated in this disease. Here we demonstrate that BLAP75, a novel protein containing a putative OB-fold nucleic acid binding domain, is an integral component of BLM complexes, and is essential for their stability in vivo. Consistent with a role in BLM-mediated processes, BLAP75 colocalizes with BLM in subnuclear foci in response to DNA damage, and its depletion impairs the recruitment of BLM to these foci. Depletion of BLAP75 by siRNA also results in deficient phosphorylation of BLM during mitosis, as well as defective cell proliferation. Moreover, cells depleted of BLAP75 display an increased level of sister-chromatid exchange, similar to cells depleted of BLM by siRNA. Thus, BLAP75 is an essential component of the BLM-associated cellular machinery that maintains genome integrity.     

The Saccharomyces cerevisiae ACP2 gene encodes an essential HMG1-like protein.

The high-mobility-group (HMG) proteins, a group of nonhistone chromatin-associated proteins, have been extensively characterized in higher eucaryotic cells. To test the biological function of an HMG protein, we have cloned and mutagenized a gene encoding an HMG-like protein from the yeast Saccharomyces cerevisiae. A yeast genomic DNA library was screened with an oligonucleotide designed to hybridize to any yeast gene containing an amino acid sequence conserved in several higher eucaryotic HMG proteins. DNA sequencing and Northern (RNA) blot analysis revealed that one gene, called ACP2 (acidic protein 2), synthesizes a poly(A)+ RNA in S. cerevisiae which encodes a 27,000-molecular-weight protein whose amino acid sequence is homologous to those of calf HMG1 and HMG2 and trout HMGT proteins. Standard procedures were used to construct a diploid yeast strain in which one copy of the ACP2 gene was mutated by replacement with the URA3 gene. When this diploid was sporulated and dissected, only half of the spores were viable. About half of the nonviable spores proceeded through two or three cell divisions and then stopped dividing; the rest did not germinate at all. None of the viable spores contained the mutant ACP2 gene, thus proving that the protein encoded by ACP2 is required for cell viability. The results presented here demonstrate that an HMG-like protein has an essential physiological function.     

Purification, characterization and crystallization of ERA, an essential GTPase from Escherichia coli

ERA is an essential GTPase widely conserved in bacteria. Homologues of ERA are also present in higher eukaryotic cells. ERA is involved in bacterial cell cycle control at a point preceding cell division. In order to aid the functional investigation of ERA and to facilitate structure-function studies, we have undertaken the X-ray crystallographic analysis of this protein. Here, we report the purification and crystallization procedures and results. The purified ERA exhibits nucleotide-binding activity and GTP-hydrolytic activity. ERA is one of the very few multi-domain GTPases crystallized to date.     

Characterization of the DNA-dependent GTPase activity of T4 gene 41 protein, an essential component of the T4 bacteriophage DNA replication apparatus

The product specified by T4 bacteriophage gene 41 is known from genetic analyses to be essential for phage DNA replication in vivo. Correspondingly, the purified gene 41 protein is an essential component of an efficient in vitro DNA replication system reconstructed from seven purified T4 replication proteins; it is required both for the synthesis of short RNA primers (in conjunction with the T4 gene 61 protein) and for the rapid unwinding of the double-helical DNA template at a replication fork. The purified gene 41 protein exhibits a DNA-dependent GTPase (and ATPase) activity. In this report, we have used this associated GTPase activity as a biochemical probe for the analysis of the interactions between DNA and the 41 protein. Our results suggest that, upon binding GTP, the 41 protein monomer is induced to form a dimer, which can them form a tight complex with single-stranded DNA. Driven by the repeated hydrolysis of GTP molecules, the 41 protein dimer appears to run rapidly along the bound DNA chain. Studies with the synthetic GTP analogue, GTP gamma S, suggest that GTP hydrolysis is required for this 41 protein movement, but that it is not essential for the function of the 41 protein in RNA primer synthesis. In sum, our observations suggest that a 41 protein dimer runs along the lagging strand template at a DNA replication fork; from this position, it functions as a DNA helicase and simultaneously interacts with the T4 gene 61 protein to make the pentaribonucleotide primers which initiate Okazaki pieces at specific primer initiation sites.