In the above article, there were errors in the Supplemental Experimental Procedures and Table S4. First, in the Supplemental Experimental Procedures, the accession numbers for DNA Microarray (GSE56936) and DNA deep sequencing (DRA002233, DDBJ Sequence Read Archive) were omitted. Second, in Table S4, a few errors were made in the sequences of oligo DNAs for the ChIP-IL8 gene. Both of these changes have been now been updated online. The authors apologize for any inconvenience this may have caused.

The gene encoding a transmembrane glycoprotein LIG-1, of which the extracellular region was organized with the leucine-rich repeats and immunoglobulin-like domains, was disrupted in mice by gene targeting. LIG-1-deficient mice developed a skin change on the tail and facial area after birth. The affected skin was histologically reminiscent of the epidermis in human common skin disease 'psoriasis'. LIG-1 was expressed in basal cells of the epidermis and outer root sheath cells of hair follicles in mice. Interestingly, the LIG-1 expression was apparently down-regulated in the psoriatic lesions, suggesting that LIG-1 inversely correlates with proliferative ability of epidermal keratinocytes. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

This article discusses the applicability to a living animal of the temperature mapping method using the water proton chemical shift obtained with three-dimensional magnetic resonance spectroscopic imaging (3D-MRSI). There are several sources of error in obtaining the spectra with 3D-MRSI: signal noise, limitation in the frequency resolution due to the finite signal length, intravoxel inhomogeneity in the static magnetic field, and variation in the magnetic field due to the eddy current magnetic field. A spectral estimation method called phase deduction complex Lorentzian fitting (PD-CLF) was proposed. Numerical simulations demonstrated that this method reduces the error in the chemical shift to one third of that obtained with the simple frequency subtraction method that uses zero-padded first Fourier transformation (FFT). The temperature images obtained using 3D-MRSI with PD-CLF clearly visualized the changes and distribution of temperature in an anesthetized rat.

RNase H1 in mammalian cells is present in nuclei and mitochondria. Its absence in mitochondria results in embryonic lethality due to the failure to amplify mitochondrial DNA (mtDNA). Dual localization to mitochondria and nuclei results from differential translation initiation at two in-frame AUGs (M1 and M27) of a single mRNA. Here we show that expression levels of the two isoforms depend on the efficiency of translation initiation at each AUG codon and on the presence of a short upstream open reading frame (uORF) resulting in the mitochondrial isoform being about 10% as abundant as the nuclear form. Translation initiation at the M1 AUG is restricted by the uORF, while expression of the nuclear isoform requires reinitiation of ribosomes at the M27 AUG after termination of uORF translation or new initiation by ribosomes skipping the uORF and the M1 AUG. Such translational organization of RNase H1 allows tight control of expression of RNase H1 in mitochondria, where its excess or absence can lead to cell death, without affecting the expression of the nuclear RNase H1.

A psychrotrophic bacterium Shewanella sp. strain SIB1 was grown at 4 and 20°C, and total soluble proteins extracted from the cells were analyzed by two-dimensional polyacrylamide gel electrophoresis. Comparison of these patterns showed that the cellular content of a protein with a molecular mass of 28 kDa and an isoelectric point of four greatly increased at 4°C compared to that at 20°C. Determination of the N-terminal amino acid sequence, followed by the cloning and sequencing of the gene encoding this protein, revealed that this protein is a member of the FKBP family of proteins with an amino acid sequence identity of 56% to Escherichia coli FKBP22. This protein was overproduced in E. coli in a His-tagged form, purified, and analyzed for peptidyl-prolyl cis-trans isomerase activity.When this activity was determined by the protease coupling assay using N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide as a substrate at various temperatures, the protein exhibited the highest activity at 10°C with a k cat /K m value of 0.87 lM . When the peptidyl-prolyl cis-trans isomerase activity was determined by the RNase T 1 refolding assay at 10 and 20°C, the protein exhibited higher activity at 10°C with a k cat /K m value of 0.50 lM . These k cat /K m values are lower but comparable to those of E. coli FKBP22. We propose that a FKBP family protein is involved in cold-adaptation of psychrotrophic bacteria.

Using previously isolated Bacillus brevis strains that secrete large amounts of proteins but little protease into the medium, we have developed a host-vector system for very efficient synthesis and secretion of heterologous proteins. The multiple promoters and the signal-peptide-coding region of the MWP gene, a structural gene for one of the major cell wall proteins of B. brevis strain 47, were used to construct expression-secretion vectors. With this system, a synthetic gene for human epidermal growth factor (hEGF) was expressed efficiently and a large amount (0.24 g per liter ofculture) of mature hEGF was secreted into the medium. hEGF purified from the culture supernatant had the same NH2-terminal amino acid sequence, COOH-terminal amino acid, and amino acid composition as natural hEGF, and it was fully active in biological assays. These results, in combination with previous results, showed that mammalian proteins can be produced in active form 10-100 times more efficiently in B. brevis than has been reported in other systems.

A cDNA encoding a protein designated as LIG-1 has been cloned and characterized. A fragment of this cDNA was found previously in a screen for genes up-regulated during neural differentiation in mouse P19 embryonal carcinoma cells. Comparative sequence analysis revealed LIG-1 to be a novel integral membrane glycoprotein (1091 amino acids) containing an extracellular region (794 amino acids) with a potential signal peptide, 15 leucine-rich repeats, 3 immnunoglobulin-like domains, and 7 potential N-glycosylation sites, a transmembrane region of 23 amino acids, and a cytoplasmic region of 274 amino acids. This protein, therefore, is a new member of both the leucine-rich repeat and the immunoglobulin superfamilies. Furthermore, Northern blot and in situ hybridization analyses showed LIG-1 gene expression to be predominantly in the brain, restricted to a small subset of glial cells such as Bergmann glial cells of the cerebellum and glial cells in the nerve fiber layer of the olfactory bulb. On the basis of its structural features and expression pattern, we propose that LIG-1 functions as a cell type-specific adhesion molecule or receptor at the glial cell surface, and plays a role in the nervous system in for example neuroglial differentiation, development, and/or maintenance of neural functions where it is expressed.

When polypeptides are synthesized at ribosomes, peptide bonds are connected in trans form. In the case of peptide bonds N-terminal of the proline residues, however, some of them form cis peptide bonds in correctly folded proteins [1]. Consequently, trans-to-cis conversions of these peptide bonds (prolyl isomerizations) should occur during protein folding reactions. As demonstrated in some refolding experiments [2,3], prolyl isomerizations are relatively slow and can be the rate limiting step in protein folding reactions. The cis-trans isomerizations of peptide bonds N-terminal of the proline residues are catalyzed by peptidylprolyl cis-trans isomerases (PPIases; EC 5.2.1.8) [4]. Three structurally unrelated families of PPIases are known. They are cyclophilins, parvulins, and FK506-binding proteins (FKBPs) [5].We have previously shown that the cellular content of FKBP22 (SIB1 FKBP22) from a psychrotrophic FKBP22 from a psychrotrophic bacterium Shewanella sp. SIB1, is a dimeric protein with peptidyl prolyl cis-trans isomerase (PPIase) activity. According to homology modeling, it consists of an N-terminal domain, which is involved in dimerization of the protein, and a C-terminal catalytic domain. A long a3 helix spans these domains. An N-domain with the entire a3 helix (N-domain + ) and a C-domain with the entire a3 helix (C-domain + ) were overproduced in Escherichia coli in a His-tagged form, purified, and their biochemical properties were compared with those of the intact protein. C-domain+ was shown to be a monomer and enzymatically active. Its optimum temperature for activity (10°C) was identical to that of the intact protein. Determination of the PPIase activity using peptide and protein substrates suggests that dimerization is required to make the protein fully active for the protein substrate or that the N-domain is involved in substrate-binding. The differential scanning calorimetry studies revealed two distinct heat absorption peaks at 32.5°C and 46.6°C for the intact protein, and single heat absorption peaks at 44.7°C for N-domain + and 35.6°C for C-domain + . These results indicate that the thermal unfolding transitions of the intact protein at lower and higher temperatures represent those of C-and N-domains, respectively. Because the unfolding temperature of C-domain + is much higher than its optimum temperature for activity, SIB1 FKBP22 may adapt to low temperatures by increasing a local flexibility around the active site. This study revealed the relationship between the stability and the activity of a psychrotrophic FKBP22.Abbreviations ALPF, N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide; CD, circular dichroism; DSC, differential scanning calorimetry; FKBP, FK506-binding protein; MIP, macrophage infectivity potentiator; PPIase, peptidyl prolyl cis-trans isomerase.