A Study on the Ultrastructure and Gene Location of Hereditary Gingival Fibromatosis

Objective\ To ascertain the histological characteristics of hereditary gingival fibromatosis and the location of HGF gene. Methods\ A pedigree analysis of HGF was made. The ultrastructure of gingival overgrown tissue was observed by electron microscopy (EMS) and the location of the HGF gene defined with microsatellite markers. Results\ The HGF consisted of coarse collagen bundles and fibrocytes, epithelial cells, smooth muscle cells, etc. were abnormally arranged; the HGF locus had been mapped to chromosome 5q13 q22. Conclusion\ The gingival pathological changes resemble 'hamartoma' and the findings have implications for identification of the underlying genetic basis of HGF.

CULTURE AND REGENERATION OF POPLAR MESOPHYLL PROTOPLASTS

Populus tomentosa is one special species of poplar growing in North China. Mesophyllprotoplasts were isolated from the axenic shoots and cultured in the modified KM8p liquidmedium. Protoplast-derived cells started to divide after 7 days of culture. The frequencyof cell division reached about 20% in 10 days. The yellowish green calli grew compact andnodular after the hormone concentration of medium was adjusted. Shoot formation occurredwhen the protoplast--derived calli were transferred onto MS medium containing zeatin andIAA or NAA. The shoots rooted readily on 1/2 MS hormone--free medium.

Construction of novel tumor necrosis factor-alpha mutants with reduced toxicity and higher cytotoxicity on human tumor cells

Two tumor necrosis factor-a mutants MT1 (32Trp157Phe) and MT2 (2Lys30Ser- 32Trp157Phe) were constructed by site-directed mutagenesis. These mutants were soluble and over-expressed in E. coli. The purity of purified mutants was above 95% by serial chromatography. The results of Western blot indicated that these mutants could be cross-reactive with monoclonal antibody against native hTNF-a. Compared to parent hTNF-a, the cytotoxicity of these mutants on murine fibrosarcoma L929 cell lines reduced 4—5 orders of magnitude but was equivalent to that of native hTNF-a on human tumor cell lines. The LD50 of mutant MT1 was reduced to 0.34% of wild type and the dose of MT2 that resulted in 30% death of mice reduced to less than 1/700 that of parent hTNF-a.

SITE-SPECIFIC DELETION OF THE N-TERMINAL SEGMENTS FROM EcoRI ENDONUCLEASE USING THE POLYMERASE CHAIN REACTION

We developed a general and efficient method for directing the deletions of DNA sequences of any lengths using polymerase chain reaction (PCR). The method was based on in vitro amplification of target sequences with site-specific deletions, Klenow end-flushing and blunt-end cloning. As an example, it was used to delete the restriction gene encoding EcoRI endonuclease, resulting in plasmids expressing two truncated forms. Assays using SDS-PAGE and gel retardation revealed the important role of the amphipathic helix (29-43) of the EcoRI endonuclease in binding to its cognate substrate.

Study of rat neuronal genes with ordered differential display method

Ordered differential display (ODD) was developed recently and has been applied to systematic comparison of expression profiles of genes. It was further improved with the specific complexing property between biotin and streptavidin by the authors. First, random primer and biotinylated oligo (dT) primer were used to make pools of double strand cDNA. Second, streptavidin-coated PCR tube is used to absorb 3′ESTs specifically to avoid the negative effect of other DNA fragments. In the case of 3′ESTs comparison patterns between embryonic brain and body of SD rat, more than forty differentially expressed genes were cloned and identified. The function of rZIC gene, one of the genes identified and cloned, was studied through ethological experiments. The result showed that rZIC gene was associated with locomotion activity of adult mice.

AN EFFICIENT SITE-DIRECTED MUTAGENESIS USING POLYMERASE CHAIN REACTION

We report here a simple and efficient method for site-directed mutagenesis using polymer-ase chain reaction (PCR). In constructing a new expression plasmid for the EcoRI restrictiongene, we made two point mutations. While one created a new Sall site prior to the SDsequence, the other replaced Glu144 with Lys. A 1.5 kb Sall-PstI fragment isolated frompER101 was used as the template. Two 25 mer oligonucleotide primers containing the de-sired mutations were synthesized and used to direct PCR amplification with Taq DNA poly-merase. About 0.5μg of the 0.49 kb fragment was obtained from 0.05 μ of the 1.5 kb frag-ment by carrying out polymerase chain reaction for 30 cycles. As calculated theoretically,99% of the product contained the desired mutations. The product was cloned into pUC19using Sall and PstI, two of the transformed colonies were randomly chosen for sequence anal-ysis, and both of them were shown to contain the desired mutations. Finally, the amplifiedfragment was cloned into pER304 to place