Naive embryonic stem cells hold great promise for research and therapeutics as they have broad and robust developmental potential. While such cells are readily derived from mouse blastocysts it has not been possible to isolate human equivalents easily, although human naive-like cells have been artificially generated (rather than extracted) by coercion of human primed embryonic stem cells by modifying culture conditions or through transgenic modification. Here we show that a sub-population within cultures of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) manifests key properties of naive state cells. These naive-like cells can be genetically tagged, and are associated with elevated transcription of HERVH, a primate-specific endogenous retrovirus. HERVH elements provide functional binding sites for a combination of naive pluripotency transcription factors, including LBP9, recently recognized as relevant to naivety in mice. LBP9-HERVH drives hESC-specific alternative and chimaeric transcripts, including pluripotency-modulating long non-coding RNAs. Disruption of LBP9, HERVH and HERVH-derived transcripts compromises self-renewal. These observations define HERVH expression as a hallmark of naive-like hESCs, and establish novel primate-specific transcriptional circuitry regulating pluripotency.

The genetic background of long-chain n-alkane degradation was investigated in detail in strain E1, a member of the genetically unexplored Dietzia genus. A suicide vector carrying a 518-bp alkB fragment was site-specifically integrated into the E1 chromosome, and the full alkB, as well as its chromosomal environment was sequenced after plasmid rescue experiments. Four out of the nine putative genes were strongly induced by long-chain n-alkanes in wild-type E1. ORF4 encoded a natural fusion protein consisting of an integral membrane alkane hydroxylase and a rubredoxin domain. The significance of the alkB-rub gene in n-alkane degradation was investigated in phenotypic tests, and the disruption mutant strain exhibited severely impaired growth on n-C(20) alkane carbon source. The mutation was successfully complemented with the expression of intact AlkB-Rub protein, the full-length form of which was detected by simultaneous immunoblotting. The presented data furnish the first experimental evidence of the in vivo existence of an AlkB-Rub natural fusion protein, which plays a major role in long-chain n-alkane degradation.

Proteins are necessary for cellular growth. Concurrently, however, protein production has high energetic demands associated with transcription and translation. Here, we propose that activity of molecular chaperones shape protein burden, that is the fitness costs associated with expression of unneeded proteins. To test this hypothesis, we performed a genome-wide genetic interaction screen in baker's yeast. Impairment of transcription, translation, and protein folding rendered cells hypersensitive to protein burden. Specifically, deletion of specific regulators of the Hsp70-associated chaperone network increased protein burden. In agreement with expectation, temperature stress, increased mistranslation and a chemical misfolding agent all substantially enhanced protein burden. Finally, unneeded protein perturbed interactions between key components of the Hsp70-Hsp90 network involved in folding of native proteins. We conclude that specific chaperones contribute to protein burden. Our work indicates that by minimizing the damaging impact of gratuitous protein overproduction, chaperones enable tolerance to massive changes in genomic expression.

The metD D-methionine transporter locus of Escherichia coli was identified as the abc-yaeE-yaeC cluster (now renamed metNIQ genes). The abc open reading frame is preceded by tandem MET boxes bracketed by the ؊10 and ؊35 boxes of a promoter. The expression driven by this promoter is controlled by the MetJ repressor and the level of methionine.D-Methionine is an effective methionine source for Escherichia coli (5,11,14). The transport system reported to take up D-methionine in E. coli is encoded by the metD locus (11, 12). The system was found to be energized by ATP and regulated by the level of the internal methionine pool (10,11,13). The metD locus was mapped between the fhuA (previously called tonA) and the proA loci (12). The specific genes involved in D-methionine transport have not yet been reported.We have identified the abc-yaeE-yaeC gene cluster (now renamed metNIQ genes) as a likely candidate for the metD locus in the fhuA-proA region. The abc gene was previously found in a search for ABC transporter ATP-binding domains (1). The PROSITE program (6) indicated that the Abc protein harbors an ATP-and GTP-binding site motif A (P-loop) (24) and an ABC transporter family signature (4). Gene yaeE encodes a putative membrane protein with a high sequence similarity to several bacterial amino acid transporters and contains a binding protein-dependent transport system inner membrane component signature (6,25). The abc and yaeE open reading frames (ORFs) overlap by 8 nucleotides. The yaeC ORF is located 39 nucleotides downstream of the yaeE stop codon and was found to possess a probable signal sequence as well as a prokaryotic membrane lipoprotein lipid attachment site (6,8), suggesting that it could be a periplasmic amino acid-binding protein.A consensus MET box (5Ј-AGACGTCT-3Ј), the binding site of the MetJ repressor (2, 20, 22), was identified upstream of abc (28). There is a 62.5% consensus MET box next to the 100% box (Fig. 1). Recently, a conformational model-based prediction also identified the MetJ-binding site upstream of abc (15). This suggested that the abc-yaeE-yaeC cluster might be part of the MET regulon.Using a neural network promoter prediction algorithm (http://searchlauncher.bcm.tmc.edu/seq-search/gene-search .html) (21), a very likely 70 promoter was predicted upstream of abc (positions 2690 to 2726, reverse strand of the sequence registered under GenBank accession no. AE000129 [3]). Its spacer region between the Ϫ35 and Ϫ10 boxes is almost completely made up of the tandem MET boxes (Fig. 1). Even the Ϫ10 box of the putative promoter is part of a third, 50% MET box.Uptake of D-methionine. To determine whether the abcyaeE-yaeC putative ABC transporter gene cluster was involved in the ability of D-methionine to satisfy a methionine requirement, we deleted the cluster. The genomic region of the wildtype E. coli K-12 strain MG1655 corresponding to positions 90 to 2643 of the sequence with GenBank accession no. AE000129 was replaced with the kanamycin resistance cassette from pUC4K (Pharmacia) by using...