Describing ancient horizontal gene transfers at the nucleotide and gene levels by comparative pathogenicity island genometrics

MOTIVATION: Lateral gene transfer is a major mechanism contributing to bacterial genome dynamics and pathovar emergence via pathogenicity island (PAI) spreading. However, since few of these genomic exchanges are experimentally reproducible, it is difficult to establish evolutionary scenarios for the successive PAI transmissions between bacterial genera. Methods initially developed at the gene and/or nucleotide level for genomics, i.e. comparisons of concatenated sequences, ortholog frequency, gene order or dinucleotide usage, were combined and applied here to homologous PAIs: we call this approach comparative PAI genometrics. RESULTS: YAPI, a Yersinia PAI, and related islands were compared with measure evolutionary relationships between related modules. Through use of our genometric approach designed for tracking codon usage adaptation and gene phylogeny, an ancient inter-genus PAI transfer was oriented for the first time by characterizing the genomic environment in which the ancestral island emerged and its subsequent transfers to other bacterial genera.     

Mammalian cell gene expression: protein glycosylation.

Considerable advances have been made in identifying the factors determining the glycosylation pattern of glycoproteins secreted by mammalian cells. This has allowed a greater appreciation of the way in which recombinant proteins may be glycosylated after expression in a heterologous system. The studies reviewed herein extend the wider view that glycosylation of native and recombinant proteins is a complex event dependent on the protein moiety, the host cell, and also the environment in which transfected cells are cultured. The details of the way in which these factors combine to establish the glycosylation pattern of a secreted protein are now beginning to be unravelled.     

哺乳类细胞基因表达系统

真核基因的转录和转译调控是哺乳类细胞基因表达系统建立和发展的基础,外源基因的表达水平不仅决定于启动子/增强子的强弱,还与剪接信号、终止信号和poly(A)信号以及质粒的拷贝数等因素有关。另外,在基因治疗的研究中,也已寻找到多种具有组织或肿瘤特异性的启动子,来达到特异性肿瘤基因治疗的目的。     

Modelling gene networks at different organisational levels

Approaches to modelling gene regulation networks can be categorized, according to increasing detail, as network parts lists, network topology models, network control logic models, or dynamic models. We discuss the current state of the art for each of these approaches. There is a gap between the parts list and topology models on one hand, and control logic and dynamic models on the other hand. The first two classes of models have reached a genome-wide scale, while for the other model classes high throughput technologies are yet to make a major impact.     

Inferring gene expression dynamics from reporter protein levels

We present a mathematical method for inferring the dynamics of gene expression from time series of reporter protein assays and cell populations. We show that estimating temporal expression dynamics from direct visual inspection of reporter protein data is unreliable when the half-life of the protein is comparable to the time scale of the expression dynamics. Our method is simple and general because it is designed only to reconstruct the pattern of protein synthesis, without assuming any specific regulatory mechanisms. It can be applied to a wide range of cell types, patterns of expression, and reporter systems, and is implemented in publicly available spreadsheets. We show that our method is robust to a several possible types of error, and argue that uncertainty about the decay kinetics of reporter proteins is the limiting factor in reconstructing the temporal pattern of gene expression dynamics from reporter protein assays. With improved estimates of reporter protein decay rates, our approach could allow for detailed reconstruction of gene expression dynamics from commonly used reporter protein systems.     

Adenosine deaminase polymorphisms at the protein and DNA levels

Adenosine deaminase (ADA, E.C. 3.5.4.4) exhibits a well-known polymorphism at the protein level. We have studied ADA and an STR polymorphism exhibiting variation of a TTTA repeat motif at intron 3 of the ADA gene in random samples from northern Portugal (N = 218) and southwestern Germany (N = 114). The ADA phenotype distribution and population data on the worldwide distribution of ADA favor recurrent mutation as an explanation for the maintenance of the ADA*2 gene product at polymorphic frequencies.     

Purification and comparative studies of alcohol dehydrogenases

Alcohol dehydrogenases from various animal and plant sources were purified by a common procedure which employed DEAE, Sephadex-G100 and affinity chromatographies. The procedure achieves an 80-130 fold purification for animal enzymes. However, only a 5-15 fold purification for plant enzymes was attained because of the instability of these enzymes. Purified alcohol dehydrogenases from animal and plant sources differ in coenzyme and substrate specificities. The enzymes from mammalian, avian and fish livers display aldehyde oxidizing and esterolytic activities in addition to alcohol oxidizing activity. However, the enzymes from plants and yeast show only the oxidative activity toward alcohols. Chemical modifications have been performed to identify amino acid residues which are essential to the oxidative and esterolytic activities of alcohol dehydrogenases.     

Mammalian myristoyl CoA: protein N-myristoyltransferase

Myristoyl CoA:Protein N-myristoyltransferase (NMT) is the enzyme which catalyses the covalent transfer of myristate from myristoyl CoA to the amino-terminal glycine residue of protein substrates. Although NMT is ubiquitous in eukaryotic cells, the enzyme levels and cellular distribution vary among tissues. In this article, we describe the properties of mammalian NMT(s) with reference to subcellular distribution, molecular weights, substrate specificity and the possible involvement of NMT in pathological processes. The cytosolic fraction of bovine brain contains multiple forms of NMT activity whereas bovine spleen contains only a single form. In bovine brain and spleen, the cytosol contained majority of NMT activity. In contrast, rabbit colon and rat liver NMT activity was predominantly particulate. Regional differences in NMT activity have been observed in both rabbit intestine and bovine brain. Results from our laboratory along with the existing knowledge, provide evidence for the existence of tissue specific isozymes of NMT.     

Mammalian protein serine/threonine phosphatase 2C: cDNA cloning and comparative analysis of amino acid sequences.

Complementary DNA encoding rat protein phosphatase 2C alpha was obtained from a liver library and used to isolate the homologous cDNAs from rabbit liver and human teratocarcinoma libraries. The amino acid sequences of the three enzymes deduced from the cDNA (382 amino acids) were extremely similar (greater than 99% identity), the maximum number of differences (between rat and human) being four. Amino acid sequences of peptides corresponding to 238 residues (61%) of the protein phosphatase 2C beta isoform from rabbit skeletal muscle were determined and showed 12 differences from the recently published sequence of the rat liver enzyme deduced from the cDNA (95% identity).