Cloning the Tra1 region of RP1

The Tra1 region of RP1 from a derivative with Tn7 inserted into the kanamycin resistance determinant was cloned, using EcoRI, into the multicopy vector plasmid pBR325. For one orientation of the cloned fragment the resultant chimeric plasmid was very frequently lost from the cell, but in the other orientation it was much more stable and also compatible with RP1. Complementation by the stable chimeric plasmid, pED800, of a series of RP1 tra mutants showed that the mutations of all those retaining sensitivity to the P-specific phages PRR1, Pf3, and PR4, or only to PR4, mapped in the Tra1 region, while only 2 out of 20 amber mutations leading to full P-specific phage-resistance did so.     

The nanomechanics of polycystin-1 extracellular region

Recent evidence suggests that polycystin-1 (PC1) acts as a mechanosensor, receiving signals from the primary cilia, neighboring cells, and extracellular matrix and transduces them into cellular responses that regulate proliferation, adhesion, and differentiation that are essential for the control of renal tubules and kidney morphogenesis. PC1 has an unusually long extracellular region ( approximately 3000 amino acids) with a multimodular structure. Proteins with a similar architecture have structural and mechanical roles. Based on the structural similarities between PC1 and other modular proteins that have elastic properties we hypothesized that PC1 functions mechanically by providing a flexible and elastic linkage between cells. Here we directly tested this hypothesis by analyzing the mechanical properties of the entire PC1 extracellular region by using single molecule force spectroscopy. We show that the PC1 extracellular region is highly extensible and that this extensibility is mainly caused by the unfolding of its Ig-like domains. Stretching the native PC1 extracellular region results in a sawtooth pattern with equally spaced force peaks that have a wide range of unfolding forces (50-200 pN). By combining single-molecule force spectroscopy and protein engineering techniques, we demonstrate that the sawtooth pattern in native PC1 extracellular region corresponds to the sequential unfolding of individual Ig-like domains. We found that Ig-like domains refold after mechanical unfolding. Hence, the PC1 extracellular region displays a dynamic extensibility whereby the resting length might be regulated through unfolding/refolding of its Ig-like domains. These force-driven reactions may be important for cell elasticity and the regulation of cell signaling events mediated by PC1.     

Monoclonal antibodies specific for adenovirus early region 1A proteins: extensive heterogeneity in early region 1A products

Hybridomas secreting monoclonal antibodies specific for the adenovirus early region 1A (E1A) proteins were prepared from BALB/c mice immunized with a bacterial trpE-E1A fusion protein. This protein is encoded by a hybrid gene that joins a portion of the Escherichia coli trpE gene and a cDNA copy of the E1A 13S mRNA (Spindler et al., J. Virol. 49:132-141, 1984). Eighty-three hybridomas that secrete antibodies which recognize the immunogen were isolated and single cell cloned. Twenty-nine of these antibodies are specific for the E1A portion of the fusion protein. Only 12 of the monoclonal antibodies can efficiently immunoprecipitate E1A polypeptides from detergent lysates of infected cells. E1A polypeptides were analyzed on one-dimensional, sodium dodecyl sulfate-polyacrylamide gels and two-dimensional, isoelectric focusing polyacrylamide gels. The E1A proteins that are specifically immunoprecipitated by the monoclonal antibodies are heterogeneous in size and charge and can be resolved into approximately 60 polypeptide species. This heterogeneity is due not only to synthesis from multiple E1A mRNAs, but also at least in part to post-translational modification. Several of the monoclonal antibodies divide the E1A polypeptides into immunological subclasses based on the ability of the antibodies to bind to the antigen. In particular, two of the monoclonal antibodies bind to the polypeptides synthesized from the 13S E1A mRNA, but not to other E1A proteins.     

Recombination at Prunus S-locus region SLFL1 gene

In Prunus, the self-incompatibility (S-) locus region is <70 kb. Two genes-the S-RNase, which encodes the functional female recognition component, and the SFB gene, which encodes the pollen recognition component-must co-evolve as a genetic unit to maintain functional incompatibility. Therefore, recombination must be severely repressed at the S-locus. Levels of recombination at genes in the vicinity of the S-locus have not yet been rigorously tested; thus it is unknown whether recombination is also severely repressed at these loci. In this work, we looked at variability levels and patterns at the Prunus spinosa SLFL1 gene, which is physically close to the S-RNase gene. Our results suggest that the recombination level increases near the SLFL1 coding region. These findings are discussed in the context of theoretical models predicting an effect of linked weakly deleterious mutations on the relatedness of S-locus specificities. Moreover, we show that SLFL1 belongs to a gene family of at least five functional genes and that SLFL1 pseudogenes are frequently found in the S-locus region.     

结核分枝杆菌RD1区研究进展

RD1区是结核分枝杆菌(MTB)在长期传代过程中丢失的重要保护性抗原,RD1区仅存在于致病性分枝杆菌中,而在卡介苗(BCG)及环境分枝杆菌中缺失。RD1区基因全长9.5kb,共有9个开放读码框,分别编码9个蛋白。RD1区是MTB毒力的关键因素之一,同时RD1区存在一种新分泌表达体系,能保证ESAT6和CFP10蛋白分泌表达。RD1区蛋白有较强的免疫原性,在MTB的预防和诊断中将可能发挥巨大作用,并有可能成为筛选抗MTB药物的理想靶抗原。     

Complementary functions of E1a conserved region 1 cooperate with conserved region 3 to activate adenovirus serotype 5 early promoters.

The amino-terminal region of the adenovirus type 5 E1a protein including conserved regions (CRs) 1 and 2 binds the 105-kDa retinoblastoma protein and a second, 300-kDa, cellular protein. We show that mutant viruses with deletions of CR1 which release the binding of either p105 or p300 still activate early promoters and infect cells productively. However, mutations which disrupt binding of both proteins disrupt early promoter activity and block the viral life cycle. Ela CR3, which has an established role in early promoter activation, can act in trans to the amino-terminal functions. This suggests that the amino terminus provides distinct, redundant functions related to p300 and Rb binding that synergize with CR3 to transactivate early genes.     

Translation efficiency of adenovirus early region 1A mRNAs deleted in the 5'' untranslated region

Adenovirus deletion mutants were studied to examine the influence of the 5' untranslated sequence on the translation of early region 1A mRNAs. Alterations of the 5' untranslated sequence, including complete deletion of the wild-type 5' untranslated sequence, did not significantly affect the rate of translation.     

The region controlling the thermosensitive effect of plasmid Rts1 on host growth is separate from the Rts1 replication region.

Rts1 is a high-molecular-weight (126 x 10(6)) plasmid encoding resistance to kanamycin. It expresses unusual temperature-sensitive phenotypes, which affect plasmid maintenance and replication, as well as host cell growth. We have cloned the essential replication region of Rts1 from pAK8, a smaller derivative which is phenotypically similar to Rts1. Restriction endonuclease digests of isolated pAK8 deoxyribonucleic acid were allowed to "self-ligate" (ligation without an additional cloning vector) and subsequently were used to transform Escherichia coli strain 20SO to kanamycin resistance. Screening of these strains for the phenotypes of thermosensitive host growth and temperature-dependent plasmid elimination demonstrated that these two properties were expressed independently. Furthermore, it was shown that the Rts1 replication locus per se is not necessarily responsible for altered host growth at the nonpermissive temperature. The kanamycin resistance fragment of pAK8 was also cloned into pBR322. Electrophoretic analysis of BamHI restriction enzyme digests of this plasmid and similar digests of an Rts1 miniplasmid has allowed the identification of an 18.6-megadalton fragment carrying the replication locus and a 14.1-megadalton fragment carrying the kanamycin resistance gene.     

UV-induced mitotic recombination in thepaba 1 region ofAspergillus nidulans

UV irradiation of diploid conidia ofAspergillus nidulans induces mitotic recombination within thepaba ₁ region. The frequency of this UV-induced intragenic recombination is enhanced if a short period of incubation in a non-selective medium precedes plating of irradiated conidia in a selective medium.     

Subdomain VIII is a specificity-determining region in MEKK1

MAPK/ERK kinase kinase 1 (MEKK1) is a mitogenactivated protein kinase kinase kinase (MAP3K) of the stress-induced JNK pathway. Once activated, MEKK1 phosphorylates the MAP2K MKK4, which in turn phosphorylates JNK. MEKK1 also has the capacity to activate IKK, the central protein kinase of the NF-kappa B pathway. The molecular determinants responsible for the ability of MEKK1 to recognize specific substrates are poorly understood. We report here that select point mutations in subdomain VIII of the protein kinase domain of MEKK1 (MEKK1 Delta) differentially affect its ability to activate MKK4 and IKK, and consequently AP1 and NF-kappa B reporter genes. Moreover, binding of MKK4 to MEKK1 Delta protects the latter from cleavage at an engineered protease target site in subdomain VIII. Collectively these results provide evidence that subdomain VIII of MEKK1 is involved not only in binding to, but also in discrimination of, protein substrates.     

Overexpression and purification of the RyR1 pore-forming region

Ryanodine receptor 1 (RyR1) is a large homotetrameric calcium channel that plays a pivotal role in skeletal muscle contraction. Sequence comparison and mutagenesis studies indicate that the pore architecture of RyR1, including the last two transmembrane helices and the luminal loop linking them, is similar to that of the bacterial KcsA K(+) channel. Here, we describe the overexpression and purification of the C-terminal polyhistidine-tagged RyR1 pore-forming region. The nonionic detergent lauryldimethylamine oxide (LDAO) was selected for solubilization of the protein based on its ability to extract the protein from the membrane and to maintain it in a monodisperse state. The protein was then purified using nickel-affinity chromatography and gel filtration. Gel filtration analysis confirmed that the RyR1 fragment containing the pore-forming region (amino acids 4829-5037) is sufficient to form a tetramer.     

Ripple-associated high-firing interneurons in the hippocampal CA1 region

By simultaneously recording the activity of individual neurons and field potentials in freely behaving mice, we found two types of interneurons firing at high frequency in the hippocampal CA1 region, which had high correlations with characteristic sharp wave-associated ripple oscillations (100―250 Hz) during slow-wave sleep. The firing of these two types of interneurons highly synchronized with ripple oscillations during slow-wave sleep, with strongly increased firing rates corresponding to individual ripple episodes. Interneuron type I had at most one spike in each sub-ripple cycle of ripple episodes and the peak firing rate was 310±33.17 Hz. Interneuron type II had one or two spikes in each sub-ripple cycle and the peak firing rate was 410±47.61 Hz. During active exploration, their firing was phase locked to theta oscillations with the highest probability at the trough of theta wave. Both two types of interneurons increased transiently their firing rates responding to the startling shake stimuli. The results showed that these two types of high-frequency interneurons in the hippocampal CA1 region were involved in the modulation of the hippocampal neural network during different states.     

Ripple-associated high-firing interneurons in the hippocampal CA1 region

By simultaneously recording the activity of individual neurons and field potentials in freely behaving mice, we found two types of interneurons firing at high frequency in the hippocampal CA1 region, which had high correlations with characteristic sharp wave-associated ripple oscillations (100–250 Hz) during slow-wave sleep. The firing of these two types of interneurons highly synchronized with ripple oscillations during slow-wave sleep, with strongly increased firing rates corresponding to individual ripple episodes. Interneuron type I had at most one spike in each sub-ripple cycle of ripple episodes and the peak firing rate was 310±33.17 Hz. Interneuron type II had one or two spikes in each sub-ripple cycle and the peak firing rate was 410±47.61 Hz. During active exploration, their firing was phase locked to theta oscillations with the highest probability at the trough of theta wave. Both two types of interneurons increased transiently their firing rates responding to the startling shake stimuli. The results showed that these two types of high-frequency interneurons in the hippocampal CA1 region were involved in the modulation of the hippocampal neural network during different states.     

C-propeptide region of human pro alpha 1 type 1 collagen interacts with thioredoxin

Thioredoxin (TRX) is one of major components of thiol reducing systems. To investigate the molecular mechanism of TRX function in the lung tissue, we screened a human lung epithelial cell cDNA library for TRX-binding protein by yeast two-hybrid systems. We isolated a plasmid containing C-propeptide region of human pro alpha 1 type 1 collagen (CP-pro alpha 1(1)). CP-pro alpha 1(1) stably binds to wild type TRX but not to mutant TRX, in which redox-active cysteine residues are substituted. Failure of the interaction of mutant TRX with CP-pro alpha 1(1) was confirmed in yeast two-hybrid systems. The CP-pro alpha 1(1)/TRX interaction was increased by dithiothreitol treatment, but was markedly inhibited by hydrogen peroxide or diamide treatment. These data showed that the reducing status of TRX active site cysteine residues is important for the TRX-CP-pro alpha 1(1) interaction, indicating that collagen biosynthesis is under the regulation of TRX-dependent redox control.     

Studies on insulin/IGF-1 hybrid and IGF-1 growth-promoting functional region

Single-chain insulin/IGF-1 hybrid-[Ins/IGF-1(C)], single-chain porcine insulin precursor-(PIP), and B10Asp PIP were prepared by protein engineering. Their growth-promoting activities in mouse breast cancer cell line GR2H6 are 10, 0.2, and 2 times that of insulin, respectively, and 29%, 0.6%, and 6% of that of IGF-1, indicating that the C domain and 9Glu of IGF-1 are important for its growth-promoting activity. Given these results and previous reports, we propose that the C domain, 9Glu, and 23Phe-26Asn beta bend are involved in the growth-promoting functional region of IGF-1.