[Expression and characterization of Kringle 1-5 domains of human plasminogen].

The cDNA encoding Kringle 1-5 domains of human plasminogen (designated as K1-5), obtained from HepG2 by RT-PCR, was cloned into expression vector pHIL-S1. The recombinant plasmid pHIL-K1-5 was transformed into Pichia pastoris GS115 and the recombinant yeast was induced by methanol to express the recombinant protein. The expressed protein was purified by lysine affinity chromatography. The recombinant K1-5 inhibited the growth of bovine aortic endothelial cells (BAEC) stimulated by the basic fibroblast growth factor (bFGF), in a dosage-dependent manner, with a half maximal concentration of 14 mg/L. And rhK1-5 inhibited 47% of the BAEC migration stimulated by bFGF at the concentration of 50 mg/L. rhK1-5 also affected the cell cycle of BAEC and caused G(0)-G(1) arrest at the concentration of 14 mg/L.

[Expression and characterization of Kringle 1-4.5 domains of human plasminogen].

The cDNA encoding Kringle 1-4 and part of Kringle 5 domains of human plasminogen (K1-4.5), obtained from HepG2 by RT-PCR, was cloned into expression vector pHIL-S1. The recombinant plasmid pHIL-K1-4.5 was transformed into Pichi pastoris GS115 and the recombinant yeast was induced to express the recombinant proteins by methanol. The expressed proteins were purified by lysine affinity chromatography to a purity of 95%. The recombinant K1-4.5 inhibited the growth of bovine capillary endothelial cells (BAEC) stimulated by the basic fibroblast growth factor (bFGF), in a dosage-dependent manner with a half maximal concentration of 2 mg/L. rhK1-4.5 also inhibited 40% of the BAEC migration stimulated by bFGF in the concentration of 1 mg/L.

The function of the nuclear matrix attachment region of silkworm rDNA as an autonomously replicating sequence in plasmid and chromosomal replication origin in yeast.

Nuclear matrix attachment regions (MARs) play a crucial role in chromatin architecture, gene expression, and DNA replication. Although it is well known that yeast autonomously replicating sequences (ARSs) bind nuclear matrix and MARs also function as ARS elements in yeast, whether a heterologous MAR or ARS element acts as a replication origin in the chromosome has not been elucidated. We previously identified a MAR (rMAR) located in the nontranscribed spacer (NTS) of silkworm Attacus ricini rDNA. We report here that this rMAR contains 10 copies of ARS consensus sequence (ACS) and several DNA unwinding regions. The rMAR employs ARS activity in yeast and a rARS element locates in the 3(') region of the rMAR. Furthermore, we have also revealed that either the rMAR or the rARS element functions as a replication origin in the chromosome. Our results provide the first direct evidence to demonstrate that heterologous rMAR and rARS display chromosomal origin activity, suggesting that the chromosome structure and replication origin of rDNA reserve some common features during evolution.

Over-expression of LPTS-L in hepatocellular carcinoma cell line SMMC-7721 induces crisis.

AIM: To evaluate the function of the longer transcripts LPTS-L in hepatocellular carcinoma cell line SMMC-7721. METHODS: SMMC-7721 cells were transfected with LPTS-L expression construct and stably transfected cells were selected by G418. Multiple single clones formed and were checked for their phenotype. In the study of the effect on telomerase activity of LPTS-L in vitro, GST-LPTS-L fusion protein was expressed in E.coli and purified by glutathione-agarose column. Telomeric repeat amplification protocol (TRAP) assays were performed to study the influence of telomerase activity in SMMC-7721 cells. RESULTS: Over-expression of LPTS-L induced SMMC-7721 cells into crisis. LPTS-L could inhibit the telomerase activity in SMMC-7721 cells in vitro. CONCLUSION: LPTS-L is a potent telomerase inhibitor. Over-expression of LPTS-L can induce hepatoma cells into crisis due to the reduction of telomerase activity.

[Restin expressed in vivo suppresses the growth of tumors in nude mice].

Restin, a homologous protein of endostatin (62% homology), is the NC domain of collagen XV at C-terminal. The recombinant restin expressed in E. coli had the ability to suppress the proliferation of bovin aortic endothelial cells and cause apoptosis. In this report, mouse restin gene was fused with a sequence of human plasminogen signal peptide by PCR and cloned into eukaryotic expression vector pCDNA3. The plasmid containing restin gene was named pCDNAXV and was transfected into human hepatoma cell line Bel7404. Stable transfected clones were screened and expression of restin was confirmed by RT-PCR and Western blot. The proliferated cells were injected subcutaneusly into nude mice. The growth of tumors formed by cells transfected with restin gene was much slower than that of control group. These results indicated that the expressed restin in vivo could suppress the growth of tumor, and this suppression might be achieved by restraining angiogenesis since the restin had no effect on the proliferation of tumor cells. At the same time, this report provided a new method to investigate the effect of anti-angiogenetic proteins on the tumor growth.

Mouse restin inhibits bovine aortic endothelial cell proliferation and causes cell apoptosis.

Restin, a homologous protein of endostatin, was found by Ramchandran et al. It was the C-terminal fragment of type XV collagen. To analysis the inhibition activity of mouse restin on the proliferation of endothelial cells, the cDNA of restin was amplified from the total RNA of the mouse muscle and cloned into the prokaryotic expression plasmid pQE32. The recombinant protein was expressed in inclusion body with a yield about 60%--70% of total protein. After refolding, the purified recombinant protein specifically inhibits bovine aortic endothelial (BAE) cell proliferation stimulated by basic fibroblast growth factor (bFGF) in a dose-dependent manner, but the activity of restin was weaker than that of endostatin. Treatment of BAE cell with recombinant restin caused G(1) arrest and apoptosis in BAE cells.

[The cloning and expression of a novel mouse gene mLPTS and its subcellular localization].

A novel mouse gene mLPTS was cloned by EST assembling, RT-PCR and DNA sequencing. The gene fragment for mLPTS is 1244 bp in length, encoding a protein of 332 amino acids. The amino acid sequence of mLPTS has 78% homologue with that of LPTS gene, which is a novel liver cancer-related gene identified through positional candidate cloning stratage by our laboratory. The expression of LPTS gene was ubiquitous in normal human tissues, whereas levels appeared to be significantly reduced, or sometime undetectable in HCC cells and neoplastic tissues, and it might be involved in the negative regulation of cell proliferation. The expression of mLPTS gene was found in all mouse tissues analyzed, same with that of LPTS gene in human. There was only one transcript for mLPTS gene in mouse tissues. The phylogenetic tree was constructed through the amino acids sequence analysis and the study of the sequence homologue among different species. Next, mLPTS gene was cloned into green fluorescent protein eukarytic expression vector and then transfected into CHO cell line. The green fluorescent was mostly limited in the nucleolus, showing that the gene products of mLPTS in eukaryocytes were located in the nucleolus.

Expression of Human Angiostatin in Pichia pastoris and the Detection of Its Anti-angiogenic Activity.

Human angiostatin cDNA was amplified from human hepatoma cell line HepG2 using RT-PCR and was cloned into pPIC9K vector. Recombinant Pichia pastoris strain with 4 copies of angiostatin gene was obtained. Recombinant protein was purified by lysine-affinity Sepharose column and the finally purified angiostatin was 25 mg/L, higher than previously reported 17 mg/L. Amino acid sequence analysis revealed the identity of our protein the same with that previously reported. Recombinant angiostatin inhibited specifically the proliferation of bovine aortic endothelial cell stimulated by bFGF, with ED(50) being about 3 mg/L.

Cloning and Characterization of fup1, A Gene Highly Expressed in Hepatocellular Carcinoma.

Primary hepatocellular carcinoma(HCC) is one of the common malignant tumors in China. In our previous work, a gene named fup1(function-unknown protein 1) was isolated that was expressed differently in HCC and in normal liver. We assumed that it might be a candidate oncogene for the HCC. The fup1 gene had a ORF of 1 233 bp, encoding a protein with M(r) of 46 kD and isoelectric point of 5.48. The sequence characteristics showed its possible localization in nuclei. Northern blots showed that this gene was weakly expressed in many types of human tissues, except in the heart, implying its tissue-specific expression pattern. MTT assay of the NIH 3T3 cells transfected with this gene in the form of recombinant eukaryotic expression plasmid showed its enhancing role to cellular proliferation.

Identification of Up-regulated Genes in Rat Regenerating Liver Tissue by Suppression Subtractive Hybridization.

Suppression subtractive hybridization (SSH) technique was used to screen up-regulated genes in regenerating liver. The cDNA from rat regenerating liver tissue was used as the tester, and that from normal liver was used as the driver. After cloning, a subtraction cDNA library of 900 clones was obtained. These 900 clones were further analyzed by differential gene expression screening, and 50 clones with obvious overexpression in regenerating liver were identified. Sequencing analysis and homology searching showed that these clones represent 37 genes, 13 of them are homologous to genes known to be involved in liver regeneration 15 are known genes which are first found to be up-regulated in regenerating liver and 9 are novel genes (ESTs) which have been deposited to GenBank. RNA dot blotting analysis was used to further study the gene expression patterns. The results not only confirmed the up-regulation of these genes in regenerating liver tissue, but also showed that these genes had different expression patterns during the process of liver regeneration. The results implied that these genes might play important roles in liver regeneration.

Regulation of Gene Expression by SAR of Silkworm Attacus ricini rRNA Gene.

The 1 kb scaffold-attachment region (SAR) at 5' non-transcription region of rRNA gene of silkworm Attacus ricini was cloned into eukaryotic expression vector pLu, which contained luciferase report gene and neo(R) selecting marker. After transfection of constructs into NIH3T3 cell line by using cation liposome, the luciferase activity was monitored to check the SAR's function. The results demonstrated that the SAR could enhance gene expression up to 15-fold in stable transformed cells, but no obvious gene expression was observed in transient transfection. Its effect on gene expression appeared to requirechromosomal integration. Southwestern blotting experiments showed that SAR specifically bound to nuclear matrix proteins of NIH3T3 cells. The binding with nuclear matrix may be necessary for SAR function of transcriptional enhancement.

On the Mechanism of Growth Inhibition of Epiregulin in A431 Epidermal Carcinoma Cells.

Preliminary investigation on the mechanism of the growth inhibition by recombinant epiregulin(EPI)of epidermal carcinoma cell A431 is reported. Northern blotting indicated that the mRNA level of cyclin dependent kinase(CDK)inhibitor, p21(WAF1/CIP1), was increased significantly after stimulation of the recombinant epiregulin protein. Luc reporter revealed that STAT1 could bind the promoter region of p21 in response to the EPI signal. Flow cytometry assay showed that the EPI-induced growth inhibition was not related to the apoptosis. The above results indicate that the EPI-induced cell growth inhibition might result from the STAT1-stimulated expression of p21, leading to the G1 arrest.

Detection of Lis1 Gene Frame Shift Mutation in Human Hepatocarcinoma.

GST fusion protein expression system combined with protein truncation test(PTT) protocol was used to detect gene frame shift mutation. The RT-PCR products of Lis1 genes from hepatocarcinoma samples were respectively cloned into a GST fusion protein expression vector pGEX-1, then expressed in E.coli. The results showed a truncated 33 kD fusion protein in SDS-PAGE, although the full-translated product of Lis1 gene should be of 71 kD. Sequencing revealed insertion of an A residue, causing the premature termination of translation, between the 163th and 164th nucleotide of Lis1 gene. This improved PTT assay was proved to be a fast and effective way in detecting gene frame shift mutation.

Expression of Human Epiregulin in E.coli.

Human epiregulin cDNA was amplified from the lung cancer cell line A549 using RT-PCR. After adding 6 His codon to its 3' end, it was cloned into a high efficient secretive Escherichia coli system with alkaline phosphatase promoter(phoA promoter)constructed in our lab and induced for expression. The product was purified one-step by Ni-NTA column. Amino acid sequence analysis revealed the identity of our product with that previously reported. The product showed strong proliferative effect on fibroblast cell line Balb/c3T3 and growth inhibitory effect on epithelial carcinoma cell line A431.

Functional Difference between N Domain and C Domain of hEGF and hTGF-alpha.

By exchanging the N domain and C domain of hEGF and hTGF-alpha genes by PCR, two chimeras E-TGF(EGF(1-32)-TGF-alpha(34-50))and T-EGF(TGF-alpha(1-33)-EGF(33-53))were constructed. The wild and chimeric molecules were expressed in E.coli under phoA system. The expressed hEGF, hTGF-alpha and two chimeras were purified. The EGF receptor competitive binding affinity of the four molecules was hEGF > hTGF-alpha and E-TGF > T-EGF and the cell proliferation stimulating activity of them was hTGF-alpha and E-TGF > T-EGF > hEGF. The result suggests that the N domain of hEGF and hTGF-alpha may play a major role in receptor binding activity and C domain of them may be responsible for stimulating cell proliferation.

The Autonomous Replication Function (ARS) of the Scaffold-associated Region (SAR) of Silkworm Attacus ricini rDNA in Yeast.

The one kilo-base scaffold-associated region (SAR) of silkworm Attacus ricini rRNA gene (rDNA) has been identified previously([1]). To investigate the critical sequence and the relative activity of ARS (autonomously replicating sequence), a set of restriction from covered the whole rRNA gene unit were subcloned into the nonreplicative pSKY vector. Among the seven plasmids constructed, the plasmid pSEY, having SAR, gave obvious positive replication activity in yeast as determined by the transformation efficiency. Further dissection of SAR demonstrated that plasmid pAAY, having a 0.26 kb small fragment of SAR was 40-50 fold more active then the whole SAR, while pSAY, having the remaining part of SAR, showed no activity. There were fifteen ARS consensus sequences (ACSs) within SAR being identified through sequenced alignment. It was found that only three ACSs in pAAY, located at the 3' end of SAR displayed a positive ARS activity and the remaining sequence having the other twelve ACSs functioned as a repressor. The in vitro binding assay showed that SAR from the silkworm rRNA gene bound to the yeast nuclear scaffold. These results suggest that SAR is evolutionarily conserved, and there is a close correlation between SAR and ARS activity. Detailed analysis of the positive and negative regulatory elements in SAR can be carried out based on these results.

The Effect of Partially Conserved Residues of hTGF-alpha C Domain in Its Structure and Function.

There is a highly homologous region in the C domain of the EGF family, some of its residues are semi-conserved. We constructed three hTGF-alpha mutants, hTGF-alphaV35, hTGF-alphaQ44, hTGF-alphaY45R46, by site-directed mutagenesis to replace the semi-conserved residues in the C domain of hTGF-alpha with the corresponding residues of hEGF. We observed that although the binding affinity of hEGF to hEGF receptor was about two fold that of hTGF-alpha, but the receptor binding affinity of the three mutants was respectively decreased to about 22 %, 13.4 % and 25 % compared of that of hTGF-alpha. On the other hand, the stimulating action of hEGF on NRK-49F cell proliferation was only 10 % that of hTGF-alpha, but those of the threemutants was about 4 fold, 10 fold and 5 fold more active than hTGF-alpha. Thus, the three mutants did not become more similar to hEGF in function. The functional difference between hEGF and hTGF-alpha was not simply determined by any single semi-conserved residue, but substitution at those sites in the C domain have altered the characters of hTGF-alpha sharply.

High Expression in Insect Cells of A Functional Chimeric Antibody with Specificity for HBsAg.

We have constructed a double-recombinant virus BacHL4.2 containing both the chimeric heavy- and light-chain cDNAs from monoclonal antibody of the HBsAg gene. Both murine-human chimeric antibody heavy- and light-chain were expressed in Sf9 cells infected with a double-recombinant virus BacHL4.2 or co-infected with separate heavy- and light-chain recombinant viruses. In both cases, expressed products were correctly assembled into normal H(2)L(2) immunoglobulin monomers. ELISA and functional immunoblot assay showed that the recombinant chimeric antibody exhibited a specificity for HBsAg similar to that of the parental murine monoclonal antibody OH3.

The DNA Sequence and Structural Characteristic of The 5'-Nontranscribed Spacer of Silkworm Attacus ricini rDNA.

We have found that the SacII-EcoRI fragment in the nontranscribed spacer (NTS) of silkworm Attacus ricini rDNA is a nuclear scaffold-associated region (SRA) and showed the function as the ARS element in yeast. This paper reports the sequence of this NTS region and the various characteristic potential functional motifs as analyzed by computer. It is 1 025 bp long and AT-Rich. With 9 bent DNA motifs, 10 T-boxes, 5 A-boxes motifs, 13 topoisomerase II as well as 15 ARS consensus sequences. In addition, there are several dozens of inverted repeats and ATTA/TAAT, ATTTA/TAAAT, ATATTT/AAATAT motifs commonly believed to be the binding sites of many homeodomain proteins. These motifs, concentrated in the SAR region, may play very important role in the regulation of gene transcription and replication at the chromatin level.

A Hepatitis B Virus Variant with an Ile to Ser Mutation at aa126 of HBsAg.

For the detection of HBV variants in patients vaccinated with HBV vaccine but failed to be protected, 16 children patients were studied by using the polymerase chain reaction (PCR) to amplify the HBV S gene fragment. To increase the sensitivity, a nested PCR method was used. These 10 HBV S gene fragments amplified from patients were cloned into M13mp18 phage vector and then sequenced respectively. One of them, No.19, was found to have a point mutation within a determinant coding region (nt524-nt595) of the HBsAg. There was a G at nt 531 instead of T, leading to a change of Ile to Ser at aa126 of the major HBsAg. As aa126 is located in the first loop of the two-looped conformational structure of the determinant, and the Ile to Ser at aa 126 is a drastic change, it is suggested that the antigenicity of the HBsAg might be altered and the immune failure in patient No.19 was probably related to the mutation.