Generation of human induced pluripotent stem cells from dermal fibroblasts.

The generation of patient-specific pluripotent stem cells has the potential to accelerate the implementation of stem cells for clinical treatment of degenerative diseases. Technologies including somatic cell nuclear transfer and cell fusion might generate such cells but are hindered by issues that might prevent them from being used clinically. Here, we describe methods to use dermal fibroblasts easily obtained from an individual human to generate human induced pluripotent stem (iPS) cells by ectopic expression of the defined transcription factors KLF4, OCT4, SOX2, and C-MYC. The resultant cell lines are morphologically indistinguishable from human embryonic stem cells (HESC) generated from the inner cell mass of a human preimplantation embryo. Consistent with these observations, human iPS cells share a nearly identical gene-expression profile with two established HESC lines. Importantly, DNA fingerprinting indicates that the human iPS cells were derived from the donor material and are not a result of contamination. Karyotypic analyses demonstrate that reprogramming of human cells by defined factors does not induce, or require, chromosomal abnormalities. Finally, we provide evidence that human iPS cells can be induced to differentiate along lineages representative of the three embryonic germ layers indicating the pluripotency of these cells. Our findings are an important step toward manipulating somatic human cells to generate an unlimited supply of patient-specific pluripotent stem cells. In the future, the use of defined factors to change cell fate may be the key to routine nuclear reprogramming of human somatic cells.

Sequence and phylogenetic analyses of 4 TMS junctional proteins of animals: connexins, innexins, claudins and occludins.

Connexins and probably innexins are the principal constituents of gap junctions, while claudins and occludins are principal tight junctional constituents. All have similar topologies with four alpha-helical transmembrane segments (TMSs), and all exhibit well-conserved extracytoplasmic cysteines that either are known to or potentially can form disulfide bridges. We have conducted sequence, topological and phylogenetic analyses of the proteins that comprise the connexin, innexin, claudin and occludin families. A multiple alignment of the sequences of each family was used to derive average hydropathy and similarity plots as well as phylogenetic trees. Analyses of the data generated led to the following evolutionary and functional suggestions: (1) In all four families, the most conserved regions of the proteins from each family are the four TMSs although the extracytoplasmic loops between TMSs 1 and 2, and TMSs 3 and 4 are usually well conserved. (2) The phylogenetic trees revealed sets of orthologues except for the innexins where phylogeny primarily reflects organismal source, probably due to a lack of relevant organismal sequence data. (3) The two halves of the connexins exhibit similarities suggesting that they were derived from a common origin by an internal gene duplication event. (4) Conserved cysteyl residues in the connexins and innexins may point to a similar extracellular structure involved in the docking of hemichannels to create intercellular communication channels. (5) We suggest a similar role in homomeric interactions for conserved extracellular residues in the claudins and occludins. The lack of sequence or motif similarity between the four different families indicates that, if they did evolve from a common ancestral gene, they have diverged considerably to fulfill separate, novel functions. We suggest that internal duplication was a general evolutionary strategy used to generate new families of channels and junctions with unique functions. These findings and suggestions should serve as guides for future studies concerning the structures, functions and evolutionary origins of junctional proteins.

Influence of threonine exporters on threonine production in Escherichia coli.

Threonine production in Escherichia coli threonine producer strains is enhanced by overexpression of the E. coli rhtB and rhtC genes or by heterologous overexpression of the gene encoding the Corynebacterium glutamicum threonine excretion carrier, thrE. Both E. coli genes give rise to a threonine-resistant phenotype when overexpressed, and they decrease the accumulation of radioactive metabolites derived from [(14)C] L-threonine. The evidence presented supports the conclusion that both RhtB and RhtC catalyze efflux of L-threonine and other structurally related neutral amino acids, but that the specificities of these two carriers differ substantially.

An integrated Arabidopsis annotation database for Affymetrix Genechip data analysis, and tools for regulatory motif searches.

Genome-scale sequencing projects have provided the essential information required for the construction of entire genome chips or microarrays for RNA expression studies. The Arabidopsis and rice genomes have been sequenced and whole-genome oligonucleotide arrays are being manufactured. These should soon become available to researchers. Expression studies using genomic-scale expression arrays are providing us with a vast quantity of information at a rapid pace. The rate-limiting step in this type of experiments is not the data generation step but rather the data analysis component of experiments. We report improvements that should facilitate the analysis of Affymetrix Genechip expression data.

Phylogenetic relationships within cation transporter families of Arabidopsis.

Uptake and translocation of cationic nutrients play essential roles in physiological processes including plant growth, nutrition, signal transduction, and development. Approximately 5% of the Arabidopsis genome appears to encode membrane transport proteins. These proteins are classified in 46 unique families containing approximately 880 members. In addition, several hundred putative transporters have not yet been assigned to families. In this paper, we have analyzed the phylogenetic relationships of over 150 cation transport proteins. This analysis has focused on cation transporter gene families for which initial characterizations have been achieved for individual members, including potassium transporters and channels, sodium transporters, calcium antiporters, cyclic nucleotide-gated channels, cation diffusion facilitator proteins, natural resistance-associated macrophage proteins (NRAMP), and Zn-regulated transporter Fe-regulated transporter-like proteins. Phylogenetic trees of each family define the evolutionary relationships of the members to each other. These families contain numerous members, indicating diverse functions in vivo. Closely related isoforms and separate subfamilies exist within many of these gene families, indicating possible redundancies and specialized functions. To facilitate their further study, the PlantsT database (http://plantst.sdsc.edu) has been created that includes alignments of the analyzed cation transporters and their chromosomal locations.

The complete phosphotransferase system in Escherichia coli.

We here tabulate and describe all currently recognized proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) and their homologues encoded within the genomes of sequenced E. coli strains. There are five recognized Enzyme I homologues and six recognized HPr homologues. A nitrogen-metabolic PTS phosphoryl transfer chain encoded within the rpoN and ptsP operons and a tri-domain regulatory PTS protein encoded within the dha (dihydroxyacetone catabolic) operon, probably serve regulatory roles exclusively. In addition to several additional putative regulatory proteins, there are 21 (and possibly 22) recognized Enzyme II complexes. Of the 21 Enzyme II complexes, 7 belong to the fructose (Fru) family, 7 belong to the glucose (Glc) family, and 7 belong to the other PTS permease families. All of these proteins are briefly described, and phylogenetic data for the major families are presented.

Genes involved in control of galactose uptake in Lactobacillus brevis and reconstitution of the regulatory system in Bacillus subtilis.

The heterofermentative lactic acid bacterium Lactobacillus brevis transports galactose and the nonmetabolizable galactose analogue thiomethyl-beta-galactoside (TMG) by a permease-catalyzed sugar:H(+) symport mechanism. Addition of glucose to L. brevis cells loaded with [(14)C]TMG promotes efflux and prevents accumulation of the galactoside, probably by converting the proton symporter into a uniporter. Such a process manifests itself physiologically in phenomena termed inducer expulsion and exclusion. Previous evidence suggested a direct allosteric mechanism whereby the phosphocarrier protein, HPr, phosphorylated at serine-46 [HPr(Ser-P)], binds to the galactose:H(+) symporter to uncouple sugar transport from proton symport. To elucidate the molecular mechanism of inducer control in L. brevis, we have cloned the genes encoding the HPr(Ser) kinase, HPr, enzyme I, and the galactose:H(+) symporter. The sequences of these genes were determined, and the relevant phylogenetic trees are presented. Mutant HPr derivatives in which the regulatory serine was changed to either alanine or aspartate were constructed. The cloned galP gene was integrated into the chromosome of Bacillus subtilis, and synthesis of the mutant HPr proteins in this organism was shown to promote regulation of GalP, as expected for a direct allosteric mechanism. We have thus reconstituted inducer control in an organism that does not otherwise exhibit this phenomenon. These results are consistent with the conclusion that inducer exclusion and expulsion in L. brevis operates via a multicomponent signal transduction mechanism wherein the presence of glycolytic intermediates such as fructose 1,6-bisphosphate (the intracellular effector), derived from exogenous glucose (the extracellular effector), activates HPr(Ser) kinase (the sensor) to phosphorylate HPr on Ser-46 (the messenger), which binds to the galactose:H(+) symporter (the target), resulting in uncoupling of sugar transport from proton symport (the response). This cascade allows bacteria to quickly respond to changes in external sugar concentrations. Understanding the molecular mechanism of inducer control advances our knowledge of the link between metabolic and transport processes in bacteria.

Phylogenetic analyses of the constituents of Type III protein secretion systems.

Multicomponent Type III protein secretion systems transfer gram-negative bacterial virulence factors directly from the bacterial cytoplasm to the cytoplasm of a host eukaryotic cell in a process that may involve a single energy-coupled step. Extensive evidence supports the conclusion that the genetic apparatuses that encode these systems have been acquired independently by different gram-negative bacteria, presumably by lateral transfer. In this paper we conduct phylogenetic analyses of currently sequenced constituents of these systems and their homologues. The results reveal the relative relatedness of these systems and show that they evolved with little or no exchange of constituents between systems. This fact suggests that horizontal transmission of the genes encoding these systems always occurred as a unit without the formation of hybrid gene clusters. Moreover, homologous flagellar proteins show phylogenetic clustering that suggests that the flagellar systems and Type III protein secretory systems diverged from each other following very early duplication of a gene cluster sharing many (but not all) genes. Phylogenies of most or all of the flagellar proteins follow those of the source organisms with little or no lateral gene transfer suggesting that homologous flagellar proteins are true orthologues. We suggest that the flagellar apparatus was the evolutionary precursor of Type III protein secretion systems.

A broad-specificity multidrug efflux pump requiring a pair of homologous SMR-type proteins.

The Bacillus subtilis genome encodes seven homologues of the small multidrug resistance (SMR) family of drug efflux pumps. Six of these homologues are paired in three distinct operons, and coexpression in Escherichia coli of one such operon, ykkCD, but not expression of either ykkC or ykkD alone, gives rise to a broad specificity, multidrug-resistant phenotype including resistance to cationic, anionic, and neutral drugs.