Peptide and glycopeptide dendrimers. Part I

Recent progress in peptide and glycopeptide chemistry make the preparation of peptide and glycopeptide dendrimers of acceptable purity, with designed structural and immunochemical properties reliable. New methodologies using unprotected peptide building blocks have been developed to further increase possibilities of their design and improve their preparation and separation. Sophisticated design of peptide and glycopeptide dendrimers has led to their use as antigens and immunogens, for serodiagnosis and other biochemical uses including drug delivery. Dendrimers bearing peptide with predetermined secondary structures are useful tools in protein de novo design. This article covers synthesis and applications of multiple antigen peptides (MAPs), multiple antigen glycopeptides (MAGs), multiple antigen peptides based on sequential oligopeptide carriers (MAP‐SOCs), glycodendrimers and template‐assembled synthetic proteins (TASPs). Part I deals with the development of various structural forms of MAPs as well as their application as antigens, immunogens, and for immunodiagnostic and biochemical purposes. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Unusual genetic phenomena associated with Tn5 mutagenesis in Alcaligenes eutrophus strain H1

Tn5 was introduced into Alcaligenes eutrophus strain H1 by a suicide vector pSUP1011. Physical characterization of mutants obtained after Tn5 mutagenesis revealed a relatively high frequency of plasmid curing, or deletion of a 50 kb plasmid DNA segment. Results of Southern hybridization and chromosomal walking indicate that the same continuous stretch of plasmid DNA (designated as D region of plasmid) is deleted in four independent isolates. Moreover, the same deletion of plasmid DNA is also observed in a mitomycin C-generated mutant strain H1-4.Journal Paper No. J-12095 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 2607, supported in part by a grant from the Iowa High Technology Council     

转座子Tn5096对刺孢吸水链霉菌北京变种RF220转座的诱变

用大肠杆菌/链霉菌穿梭质粒pCZA168(bla,tsr,Tn5096,ColEI rep,Strep repts)多次转化农抗120产生菌刺孢吸水链霉菌北京变种(S.Streptomyces hygrospinocus var. beijingensis)RF220的原生质体,均未得到转化子。来自吸水链霉菌应城变种(S.Streptomyces hygroscopicus var.)10-22突变株的链霉菌质粒pIJ702(tsr mel+)可以转化RF220,但转化频率只有数十个转化子/μgDNA。用来自RF220本身的pIJ702对消除pIJ702后的RF220的原生质体进行了再转化,转化率没有明显的提高。用氨苄青霉素和甘氨酸协同处理RF220的菌丝体,并经-70℃冷冻原生质体再转化,得到了4个pCZA168的转化子。质粒提取、酶切、抗性测定表明：4个转化子中pCZA168中大肠杆菌DNA部分均被切除,成为大小约50～60kb的小质粒,命名为pWZH102(tsr,Tn5096,strep repts)。用pWZH102上的转座子Tn5096对RF220进行转座实验,在168个转座个体中,有2株可能为抗生素生物合成阻断变株,另有产生抗生素水平各异的变株,说明Tn5096的转座可以引起表型的不同变化。     

运用生色基因标记黄瓜根围促生菌(PGPR)筛选菌株

采用三亲交配方法 ,通过Tn7转座系统将lacZY标记基因导入黄瓜根围促生菌 (PG PR)筛选菌株PseudomonasfluorescensCN1 1 6和PseudomonascorrugataCN31的利福平抗性突变株中 ;标记假单胞菌菌株则被赋予了利用乳糖作为唯一碳源的能力 ,在只有乳糖的M9培养基上生长能分解X Gal,菌落显出特有的蓝色 ;经Southern杂交分析 ,证明标记基因lacZY存在于转化菌株的染色体上 ;经验证标记菌株标记性状稳定 ,与对应的野生菌株比较其它性状如培养性状、形态特征、生防效果等基本不变 ;PGPR菌株利福平抗性和生色基因标记的结合 (双标记 )能最大限度地将土壤中引入的PGPR菌株与土著细菌分开 ,检测下限可达 1 0CFU mL ,为PGPR在根围的分子生态学研究提供了一个较好的工具。     

Effects of the cardiomyopathy-causing E244D mutation of troponin T on the structures of cardiac thin filaments studied by small-angle X-ray scattering

Small-angle X-ray scattering experiments were carried out to investigate the structural changes of cardiac thin filaments induced by the cardiomyopathy-causing E244D mutation in troponin T (TnT). We examined native thin filaments (NTF) from a bovine heart, reconstituted thin filaments containing human cardiac wild-type Tn (WTF), and filaments containing the E244D mutant of Tn (DTF), in the absence and presence of Ca²⁺. Analysis by model calculation showed that upon Ca²⁺-activation, tropomyosin (Tm) and Tn in the WTF and NTF moved together in a direction to expose myosin-binding sites on actin. On the other hand, Tm and Tn of the DTF moved in the opposite directions to each other upon Ca²⁺-activation. These movements caused Tm to expose more myosin-binding sites on actin than the WTF, suggesting that the affinity of myosin for actin is higher for the DTF. Thus, the mutation-induced structural changes in thin filaments would increase the number of myosin molecules bound to actin compared with the WTF, resulting in the force enhancement observed for the E244D mutation.     

Nucleotide sequence of the Streptococcus faecalis plasmid gene encoding the 3'5"-aminoglycoside phosphotransferase type III

We have cloned in Escherichia coli and sequenced a 1489-bp DNA fragment conferring resistance to kanamycin and originating from the streptococcal plasmid pJH1. The resistance gene was located by analysis of the initiation and termination codons in an open reading frame (ORF) of 792 bp. The deduced gene product, a 3'5'-aminoglycoside phosphotransferase of type III, has an Mr of 29,200. Comparison of its amino acid sequence with those of type I (Oka et al., 1981) and type II (Beck et al., 1982) 3' phosphotransferase, from transposable elements Tn903 and Tn5, respectively, indicated a statistically significant structural relationship between these enzymes from phylogenetically remote bacterial genera. The degree of homology observed indicate that phosphotransferase type III and type I genes have diverged from a common ancestor and that the phosphotransferase type II gene has emerged more recently from the type I evolutionary pathway.     

Lethality of palindromic DNA and its use in selection of recombinant plasmids

A plasmid derived from ColE1 is constructed so that the removal of one restriction endonuclease HindIII fragment allows the ends of the remaining single fragment (the replicator) to be joined, generating a palindromic sequence 2394 bp in length. The circular species thus produced gives rise to transformants of E. coli at very low frequency. Since the palindromic sequence is effectively lethal to a plasmid containing it, the replicator will give rise to more transformants when the restriction fragment originally removed from it is replaced by another. This principle can be exploited to allow the efficient molecular cloning of unselected restriction fragments.     

华癸中生根瘤菌共生质粒的定向标记、消除与结瘤功能的鉴定

采用Tn5-mob-sacB转座子对华癸中生根瘤菌(Mesorhizobium huakuii)菌株7653R的共生质粒进行定向标记,获得该质粒标记菌株7653RT14.利用sacB基因对蔗糖的敏感性,对标记质粒进行消除实验,获得7653R的共生质粒消除突变株7653R-1.测得Tn5-mob-sacB转座频率高于10-5.突变株的培养特征与出发菌株基本一致.采用琼脂管法对7653RT14和7653R-1进行回接实验,结果显示7653RT14能正常结瘤固氮,表明Tn5的插入并未影响其共生能力,但失去共生质粒的7653R-1则为不结瘤或只结个别小瘤.稳定性实验结果表明供试菌株的标记质粒在本实验条件下是稳定的,可以作为共生质粒转移的供体菌.     

Tn5 insertion mutants of Pseudomonas sp. 267 defective in siderophore production and their effect on clover (Trifolium pratense) nodulated with Rhizobium leguminosarum bv. trifolii

Pseudomonas sp. strain 267 isolated from soil promoted growth of different plants under field conditions and enhanced symbiotic nitrogen fixation in clover under gnotobiotic conditions. This strain produced pyoverdine-like compound under low-iron conditions and secreted vitamins of the B group. The role of fluorescent siderophore production in the beneficial effect of strain 267 on nodulated clover plants was investigated. Several non-fluorescent (Pvd^-) Tn5 insertion mutants of Pseudomonas sp. strain 267 were isolated and characterized. The presence of Tn5 insertions was confirmed by Southern analysis of EcoRI digested genomic DNA of each derivative strain. The siderophore-negative mutants were compared to the parental strain with respect to their growth promotion of nodulated clover infected with Rhizobium leguminosarum bv. trifolii 24.1. We found that all isolated Pvd^- mutants stimulated growth of nodulated clover plants in a similar manner to the parental strain. No consistent differences were observed between strain 267 and Pvd^- derivatives strains with respect to their plant growth promotion activity under gnotobiotic conditions.Dr Deryto died in august 1994     

The Role of the N-Terminus of the Myosin Essential Light Chain in Cardiac Muscle Contraction

To study the regulation of cardiac muscle contraction by the myosin essential light chain (ELC) and the physiological significance of its N-terminal extension, we generated transgenic (Tg) mice by partially replacing the endogenous mouse ventricular ELC with either the human ventricular ELC wild type (Tg-WT) or its 43-amino-acid N-terminal truncation mutant (Tg-Δ43) in the murine hearts. The mutant protein is similar in sequence to the short ELC variant present in skeletal muscle, and the ELC protein distribution in Tg-Δ43 ventricles resembles that of fast skeletal muscle. Cardiac muscle preparations from Tg-Δ43 mice demonstrate reduced force per cross-sectional area of muscle, which is likely caused by a reduced number of force-generating myosin cross-bridges and/or by decreased force per cross-bridge. As the mice grow older, the contractile force per cross-sectional area further decreases in Tg-Δ43 mice and the mutant hearts develop a phenotype of nonpathologic hypertrophy while still maintaining normal cardiac performance. The myocardium of older Tg-Δ43 mice also exhibits reduced myosin content. Our results suggest that the role of the N-terminal ELC extension is to maintain the integrity of myosin and to modulate force generation by decreasing myosin neck region compliance and promoting strong cross-bridge formation and/or by enhancing myosin attachment to actin.