The DAZ gene located on the Y chromosome

The DAZ gene located on the Y chromosome and autosomal DAZ similar genes DAZL1, encodes an only in sperm cells, expression of RNA-binding protein, Drosophila male sterility gene with source, in the DAZL1 knockout mouse infertility occurs, the DAZ gene play a role in the process of spermatogenesis all mammalian homologous genes have DAZL1 autosomal DAZ gene exists only in apes and some kind of monkey two C-terminal repeat sequences are different. DAZ do bait, the yeast two-hybrid screening with whom interaction DAZ associated protein of unknown function 2 (DAZAP2), with HBcAg in liver library screening to the protein, and the two clones, suggesting that HBV infection and the development of gender differences with which HBcAg in mitochondrial nucleoprotein L41, NADH dehydrogenase subunit 4, cytochrome C oxidase, cytochrome B and so the role of support HBcAg by liver mitochondria mediated HBV replication and assembly. discovery of new genes for new clues in our study HBcAg functionality, but needs further study confirmed.

 

In the control group do not have an anti-does not detect any fluorescent signa

To confirm the role of the NAC of two subunits, the expression of alpha-NAC and beta-NAC in E. coli, respectively. The use of GST-α-NAC first steps, often 70 kDa co-purified protein confirmed by Western blotting of DnaK. In addition, β-NAC is a histidine-tagged protein can be purified by this method can get more production. Carrying a histidine tag and do not carry the histidine-tagged beta-NAC and alpha-the NAC can form a functional alpha / beta complex. Re-assembled to the NAC can be purified from 10-15μm, which is purified from bovine brain NAC concentration of 3-4 times.The two subunits of the NAC and the nascent polypeptide chain on the ribosome.Can show that the combination of NAC and on the ribosome nascent polypeptide chain by the method of cross-linking reaction. Fig4. In 77aaffLuc not contain the ER signal sequence. The RNC is extracted under high salt conditions, and with the reorganization of α-NAC, histidine-tagged beta-NAC, reorganization of the NAC and NAC purified from bovine brain were incubated after UV irradiation, by SDS- PAGE and fluorescence imaging analysis. Do not add any factor after UV irradiation and no significant cross-linking. 

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NAC positions in intact cells

The NAC positioning in the cytoplasm. NAC was initially in the cytoplasm of bovine brain was isolated and purified, and the combination of ribosomes in the cytoplasm, suggesting that NAC is a cytoplasmic protein. We recently reported that, in MC3T3-E1 cells after serum starvation, immunofluorescence assay using monoclonal anti-GST-alpha-NAC serum (rather than affinity-purified antibody), alpha-NAC located in the nucleus ( 24). Therefore, we affinity-purified antibodies (anti-alpha-NAC and β-NAC-specific antibodies) positioning the NAC in intact cells. The use of different cell lines (including COS-HeLa-positioning of the Western blot of Hep-G2, and and the HL60 cell line) of the NAC. In all trials, most of the NAC is located in the cytoplasm. Printing and dyeing of MC3T3-E1 also shows that located in the cytoplasm, regardless of whether serum starvation. Cell growth to the semi-integration period. For 24h to the G0/G1 phase cells were harvested in parallel to experimental samples, serum-free conditions; or have been cultured to replace the culture medium. Most of the α-NAC and beta-NAC are clearly located in the cytoplasm. 

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Α-NAC alone can not combined with the ribosome

SDS-PAGE of α-NAC and beta-NAC separation for the two bands. Further experiments showed that the two subunits of the stoichiometric ratio of 1:1. Other methods to further support the NAC is the stoichiometric ratio 1:1 heterologous complex this hypothesis. DSP reagent results show that alpha-NAC and beta-NAC form a complex, the complex separated into two subunits in the DTT treatment. In addition, our mass spectrometry analysis of purified NAC. Received a total of three peaks, corresponding to alpha-NAC (23,492 Da) and β-NAC (17,352 Da), and they form hetero-dimer (40,864 Da). This indicates that the difference between the calculated molecular weight by SDS-PAGE molecular weight, is not due to modification, but from a special run on a gel by SDS-PAGE behavior. Detected a complete complex peak showed that in fact a very stable form.The molecular weight chromatography experiments showed that crude extracts of bovine brain NAC is also a complex form of existence.

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NAC can prevent errors combination of the nascent polypeptide chain,

In order to understand the NAC how the nascent polypeptide chain transported to the correct cotranslational acting factors, we first proposed the following questions: whether the two subunits of the NAC, respectively, to undertake specific functions; whether these two subunits is always in the form of complex exist. Our experiments show that NAC NAC as well as the crude extract obtained through a variety of purification means, in the form of the α subunit and β subunits to form a stable complex. To determine the individual role of the two subunits, and re-assembly complex in E. coli expressing human α-NAC and beta-NAC. Each subunit can be alone with ribosomes on the nascent polypeptide chain interaction.

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