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Publisher Summary This chapter discusses the regulation of isoleucine and valine biosynthesis. Isoleucine and valine biosynthesis is a model system for the study of mechanisms by which the cell coordinates different parts of its total metabolism. Threonine deaminase is the first enzyme in isoleucine biosynthesis. A different threonine deaminase, termed catabolic, appears only in anaerobiosis and in the absence of glucose. A third threonine deaminase, present in Escherichia coli K-12 and Salmonella Typhimurium, deaminates only D-threonine, and its presence explains why ilvA mutants of these organisms grow upon addition of D-threonine to the medium. The second step in the pathway is common to isoleucine and valine biosynthesis and is catalyzed by acetolactate synthase activity. It involves the conversion of either two molecules of pyruvate to form α-acetolactate, or one molecule of pyruvate and one molecule of α-ketobutyrate to form α-aceto-α-hydroxybutyrate. The acetolactate synthase isoenzymes have some relation to lysine biosynthesis. The third step in isoleucine biosynthesis and the second in valine biosynthesis are catalyzed by the enzyme isomeroreductase. The last intermediates in the pathway for isoleucine and valine biosynthesis are the keto acids from which the corresponding amino acids are synthesized by transamination.

A genomic DNA library of the rumen bacterium Streptococcus bovis was constructed in Escherichia coli, and recombinant plasmids able to complement proA and proB mutations of the host were found. Southern hybridization and restriction analysis showed that a 3.5-kb fragment of S. bovis DNA contained two genes, organized in an operon and coding for enzymes functionally similar to the glutamyl phosphate reductase-glutamyl kinase enzyme complex that in E. coli catalyzes the first step of proline biosynthesis. Complementation of the E. coli mutations was observed with the fragment inserted in both orientations, which suggested that the S. bovis proBA operon was transcribed from its own promoter. Genetic and biochemical data suggested that the proline biosynthetic pathway of S. bovis is similar to the one previously characterized for E. coli.

We developed a novel surface display system based on the use of bacterial spores. A protein of the Bacillus subtilis spore coat, CotB, was found to be located on the spore surface and used as fusion partner to express the 459-amino-acid C-terminal fragment of the tetanus toxin (TTFC). Western, dot blot and fluorescent-activated cell sorting analyses were used to monitor TTFC surface expression on purified spores. We estimated that more than 1.5 × 10 3 TTFC molecules were exposed on the surface of each spore and recognized by TTFC-specific antibodies. The efficient surface presentation of the heterologous protein, together with the simple purification procedure and the high stability and safety record of B. subtilis spores, makes this spore-based display system a potentially powerful approach for surface expression of bioactive molecules.

Spores of various Bacillus species, including B. subtilis, B. cereus and B. clausii, are used as probiotics, although they are generally absent from the normal microflora of man. We used two nonpathogenic Bacillus species, B. subtilis and B. clausii, to follow the fate of spores inoculated intragastrically in mice. We did not find detectable amounts of vegetative cells in intestinal samples, probably because of high toxicity of the conjugated bile salt taurodeoxycholic acid against Bacillus species. Both spores and cells were detected in the lymph nodes and spleen of one mouse. Our results indicate that Bacillus is present in the intestinal tract solely as spores and that nonpathogenic Bacillus spores may germinate in lymphoid organs, a finding reminiscent of B. anthracis germination in macrophages. These results indicate that any claimed probiotic effect of B. subtilis should be due to spores or, alternatively, to vegetative growth outside the intestine.

Alteration of thyroid gland morphogenesis (thyroid dysgenesis) is a frequent human malformation. Among the one in three to four thousand newborns in which congenital hypothyroidism is detected, 80% have either an ectopic, small and sublingual thyroid, or have no thyroid tissue1. Most of these cases appear sporadically, although a few cases of recurring familial thyroid dysgenesis have been described2. The lack of evidence for hereditary thyroid dysgenesis may be due to the severity of the hypothyroid phenotype. Neonatal screening and early thyroid hormone therapy have eliminated most of the clinical consequences of hypothyroidism such that the heritability of this condition may become apparent in the near future. We have recently cloned cDNA encoding a forkhead domain-containing transcription factor, TTF-2, and have located the position of the gene, designated Titf2, to mouse chromosome 4 (ref. 3). Titf2 is expressed in the developing thyroid, in most of the foregut endoderm and in craniopharyngeal ectoderm, including Rathke's pouch3. Expression of Titf2 in thyroid cell precursors is down-regulated as they cease migration, suggesting that this factor is involved in the process of thyroid gland morphogenesis. Here we show that Titf2-null mutant mice exhibit cleft palate and either a sublingual or completely absent thyroid gland. Thus, mutation of Titf2 –/– results in neonatal hypothyroidism that shows similarity to thyroid dysgenesis in humans.

The possible regulation of the expression of uncoupling protein-2 (UCP2) mRNA by thyroid hormones in different tissues was examined in rats, Triiodothyronine (T-3) was found to produce an organ-specific enhancement of UCP2 expression in rat tissues. The effect of T-3 was markedly observed in heart, whereas a moderate effect was seen in skeletal muscle and no effect in kidney or liver, These results suggest that UCP2 is a protein that may be involved in the nuclear-mediated effect of T-3 on resting metabolic rate in the rat. (C) 1997 Federation of European Biochemical Societies.

An in vivo footprinting analysis of the ilvIH regulatory region of Escherichia coli showed that the transcription activator Lrp binds to six sites, scattered over 250 bp upstream of the transcriptional start point. When Lrp-mediated activation was impaired by the presence of exogenous leucine, only one promoter-distal site (site 2) was partially protected by Lrp binding. Equilibrium dialysis experiments showed the formation of an Lrp-leucine complex in vitro. These results suggest that leucine negatively affects ilvIH transcription because its interaction with Lrp reduces the efficiency of binding of the regulatory protein to the promoter region.

Embryonic stem cells (ESCs) are derived from inner cell mass (ICM) of the blastocyst. In serum/LIF culture condition, they show variable expression of pluripotency genes that mark cell fluctuation between pluripotency and differentiation metastate. The ESCs subpopulation marked by zygotic genome activation gene (ZGA) signature, including Zscan4, retains a wider differentiation potency than epiblast-derived ESCs. We have recently shown that retinoic acid (RA) significantly enhances Zscan4 cell population. However, it remains unexplored how RA initiates the ESCs to 2-cell like reprogramming. Here we found that RA is decisive for ESCs to 2C-like cell transition, and reconstructed the gene network surrounding Zscan4. We revealed that RA regulates 2C-like population co-activating Dux and Duxbl1. We provided novel evidence that RA dependent ESCs to 2C-like cell transition is regulated by Dux, and antagonized by Duxbl1. Our suggested mechanism could shed light on the role of RA on ESC reprogramming.

Endoderm-derived organs as liver and pancreas are potential targets for regenerative therapies, and thus, there is great interest in understanding the pathways that regulate the induction and specification of this germ layer. Currently, the knowledge of molecular mechanisms that guide the in vivo endoderm specification is restricted by the lack of early endoderm specific markers. Nephrocan (Nepn) is a gene whose expression characterizes the early stages of murine endoderm specification (E7.5&ndash, 11.5) and encodes a secreted N-glycosylated protein. In the present study, we report the identification of a new transcript variant that is generated through alternative splicing. The new variant was found to have differential and tissue specific expression in the adult mouse. In order to better understand Nepn role during endoderm specification, we generated Nepn knock-out (KO) mice. Nepn&minus, /&minus, mice were born at Mendelian ratios and displayed no evident phenotype compared to WT mice. In addition, we produced nullizygous mouse embryonic stem cell (mESC) line lacking Nepn by applying (CRISPR)/CRISPR-associated systems 9 (Cas9) and employed a differentiation protocol toward endoderm lineage. Our in vitro results revealed that Nepn loss affects the endoderm differentiation impairing the expression of posterior foregut-associated markers.

Forkhead box E1 (FOXE1) is a lineage-restricted transcription factor involved in thyroid cancer susceptibility. Cancer-associated polymorphisms map in regulatory regions, thus affecting the extent of gene expression. We have recently shown that genetic reduction of FOXE1 dosage modifies multiple thyroid cancer phenotypes. To identify relevant effectors playing roles in thyroid cancer development, here we analyse FOXE1-induced transcriptional alterations in thyroid cells that do not express endogenous FOXE1. Expression of FOXE1 elicits cell migration, while transcriptome analysis reveals that several immune cells-related categories are highly enriched in differentially expressed genes, including several upregulated chemokines involved in macrophage recruitment. Accordingly, FOXE1-expressing cells induce chemotaxis of co-cultured monocytes. We then asked if FOXE1 was able to regulate macrophage infiltration in thyroid cancers in vivo by using a mouse model of cancer, either wild type or with only one functional FOXE1 allele. Expression of the same set of chemokines directly correlates with FOXE1 dosage, and pro-tumourigenic M2 macrophage infiltration is decreased in tumours with reduced FOXE1. These data establish a novel link between FOXE1 and macrophages recruitment in the thyroid cancer microenvironment, highlighting an unsuspected function of this gene in the crosstalk between neoplastic and immune cells that shape tumour development and progression.

The transcription factor Forkhead box E1 (FOXE1) is a key player in thyroid development and function and has been identified by genome-wide association studies as a susceptibility gene for papillary thyroid cancer. Several cancer-associated polymorphisms fall into gene regulatory regions and are likely to affect FOXE1 expression levels. However, the possibility that changes in FOXE1 expression modulate thyroid cancer development has not been investigated. Here, we describe the effects of FOXE1 gene dosage reduction on cancer phenotype in vivo. Mice heterozygous for FOXE1 null allele (FOXE1+/&minus, ) were crossed with a BRAFV600E-inducible cancer model to develop thyroid cancer in either a FOXE1+/+ or FOXE1+/&minus, genetic background. In FOXE1+/+ mice, cancer histological features are quite similar to that of human high-grade papillary thyroid carcinomas, while cancers developed with reduced FOXE1 gene dosage maintain morphological features resembling less malignant thyroid cancers, showing reduced proliferation index and increased apoptosis as well. Such cancers, however, appear severely undifferentiated, indicating that FOXE1 levels affect thyroid differentiation during neoplastic transformation. These results show that FOXE1 dosage exerts pleiotropic effects on thyroid cancer phenotype by affecting histology and regulating key markers of tumor differentiation and progression, thus suggesting the possibility that FOXE1 could behave as lineage-specific oncogene in follicular cell-derived thyroid cancer.

DNAJC17 is a heat shock protein (HSP40) family member, identified in mouse as susceptibility gene for congenital hypothyroidism. DNAJC17 knockout mouse embryos die prior to implantation. In humans, germline homozygous mutations in DNAJC17 have been found in syndromic retinal dystrophy patients, while heterozygous mutations represent candidate pathogenic events for myeloproliferative disorders. Despite widespread expression and involvement in human diseases, DNAJC17 function is still poorly understood. Herein, we have investigated its function through high-throughput transcriptomic and proteomic approaches. DNAJC17-depleted cells transcriptome highlighted genes involved in general functional categories, mainly related to gene expression. Conversely, DNAJC17 interactome can be classified in very specific functional networks, with the most enriched one including proteins involved in splicing. Furthermore, several splicing-related interactors, were independently validated by co-immunoprecipitation and in vivo co-localization. Accordingly, co-localization of DNAJC17 with SC35, a marker of nuclear speckles, further supported its interaction with spliceosomal components. Lastly, DNAJC17 up-regulation enhanced splicing efficiency of minigene reporter in live cells, while its knockdown induced perturbations of splicing efficiency at whole genome level, as demonstrated by specific analysis of RNAseq data. In conclusion, our study strongly suggests a role of DNAJC17 in splicing-related processes and provides support to its recognized essential function in early development.

Integrins are heterodimers anchoring cells to the surrounding extracellular matrix (ECM), an active and complex process mediating a series of inside-out and outside-in stimuli regulating cellular turn-over, tissue growth and architecture. Itgb1 is the main subunit of the renal integrins and it is critical for renal development. This study aims to investigate the role of Itgb1 in the adult renal epithelial cells by knocking down Itgb1 in PAX8 expressing cells. Itgb1-Pax8 cKO mice develop a progressively worsening proteinuria and renal abnormalities leading to severe renal failure and hypertension. This phenotype is also associated with severe dysfunction of distal nephron and polyuria. To further investigate whether distal nephron involvement was primarily related to Itgb1 suppression or secondary to renal failure, an Itgb1-AQP2 cKO mouse model was generated. These mice lack Itgb1 expression in AQP2 expressing cells. They do not show any developmental alteration, but 1 month old mice are resistant to dDAVP administration and finally, at 2 months of age, they develop overt polyuria. This phenotype is due to primary collecting duct (CD) cells anoikis. The entire architecture of the outer medulla is altered, with loss of the typical organization pattern of vascular and tubular bundles alternation. Indeed, even though not primarily affected by genetic ablation, the TAL is secondarily affected in this model. It is sufficient to suppress Itgb1 expression in the CD in order to stimulate proliferation and then disappearance of neighboring TAL cells. This study shows that cell to cell interaction through the ECM is critical for architecture and function maintenance of the outer medulla and that Itgb1 is crucial for this process. Integrins are heterodimers anchoring cells to the surrounding extracellular matrix (ECM), an active and complex process mediating a series of inside-out and outside-in stimuli regulating cellular turn-over, tissue growth and architecture. Itgb1 is the main subunit of the renal integrins and it is critical for renal development. This study aims to investigate the role of Itgb1 in the adult renal epithelial cells by knocking down Itgb1 in PAX8 expressing cells. Itgb1-Pax8 cKO mice develop a progressively worsening proteinuria and renal abnormalities leading to severe renal failure and hypertension. This phenotype is also associated with severe dysfunction of distal nephron and polyuria. To further investigate whether distal nephron involvement was primarily related to Itgb1 suppression or secondary to renal failure, an Itgb1-AQP2 cKO mouse model was generated. These mice lack Itgb1 expression in AQP2 expressing cells. They do not show any developmental alteration, but 1 month old mice are resistant to dDAVP administration and finally, at 2 months of age, they develop overt polyuria. This phenotype is due to primary collecting duct (CD) cells anoikis. The entire architecture of the outer medulla is altered, with loss of the typical organization pattern of vascular and tubular bundles alternation. Indeed, even though not primarily affected by genetic ablation, the TAL is secondarily affected in this model. It is sufficient to suppress Itgb1 expression in the CD in order to stimulate proliferation and then disappearance of neighboring TAL cells. This study shows that cell to cell interaction through the ECM is critical for architecture and function maintenance of the outer medulla and that Itgb1 is crucial for this process.

Epidemiological and experimental data highlighted the thyroid-disrupting activity of bisphenol A (BPA). Although pivotal to identify the mechanisms of toxicity, direct low-dose BPA effects on thyrocytes have not been assessed. Here, we report the results of microarray experiments revealing that the transcriptome reacts dynamically to low-dose BPA exposure, adapting the changes in gene expression to the exposure duration. The response involves many genes, enriching specific pathways and biological functions mainly cell death/proliferation or DNA repair. Their expression is only slightly altered but, since they enrich specific pathways, this results in major effects as shown here for transcripts involved in the DNA repair pathway. Indeed, even though no phenotypic changes are induced by the treatment, we show that the exposure to BPA impairs the cell response to further stressors. We experimentally verify that prolonged exposure to low doses of BPA results in a delayed response to UV-C-induced DNA damage, due to impairment of p21-Tp53 axis, with the BPA-treated cells more prone to cell death and DNA damage accumulation. The present findings shed light on a possible mechanism by which BPA, not able to directly cause genetic damage at environmental dose, may exert an indirect genotoxic activity.

To protect their genetic material cells adopt different mechanisms linked to DNA replication, recombination and repair. Several proteins function at the interface of these DNA transactions. In the present study, we report on the identification of a novel archaeal DNA helicase. BlastP searches of the Sulfolobus solfataricus genome database allowed us to identify an open reading frame (SSO0112, 875 amino acid residues) having sequence similarity with the human RecQ5β. The corresponding protein, termed Hel112 by us, was produced in Escherichia coli in soluble form, purified to homogeneity and characterized. Gel-filtration chromatography and glycerol-gradient sedimentation analyses revealed that Hel112 forms monomers and dimers in solution. Biochemical characterization of the two oligomeric species revealed that only the monomeric form has an ATP-dependent 3′–5′ DNA-helicase activity, whereas, unexpectedly, both the monomeric and dimeric forms possess DNA strand-annealing capability. The Hel112 monomeric form is able to unwind forked and 3′-tailed DNA structures with high efficiency, whereas it is almost inactive on blunt-ended duplexes and bubble-containing molecules. This analysis reveals that S. solfataricus Hel112 shares some enzymatic features with the RecQ-like DNA helicases and suggests potential cellular functions of this protein.

Epidemiologic and experimental studies have associated changes of blood glucose homeostasis to Bisphenol A (BPA) exposure. We took a toxicogenomic approach to investigate the mechanisms of low-dose (1 × 10−9M) BPA toxicity in ex vivo cultures of primary murine pancreatic islets and hepatocytes. Twenty-nine inhibited genes were identified in islets and none in exposed hepatocytes. Although their expression was slightly altered, their impaired cellular level, as a whole, resulted in specific phenotypic changes. Damage of mitochondrial function and metabolism, as predicted by bioinformatics analyses, was observed: BPA exposure led to a time-dependent decrease in mitochondrial membrane potential, to an increase of ROS cellular levels and, finally, to an induction of apoptosis, attributable to the bigger Bax/Bcl-2 ratio owing to activation of NF-κB pathway. Our data suggest a multifactorial mechanism for BPA toxicity in pancreatic islets with emphasis to mitochondria dysfunction and NF-κB activation. Finally, we assessed in vitro the viability of BPA-treated islets in stressing condition, as exposure to high glucose, evidencing a reduced ability of the exposed islets to respond to further damages. The result was confirmed in vivo evaluating the reduction of glycemia in hyperglycemic mice transplanted with control and BPA-treated pancreatic islets. The reported findings identify the pancreatic islet as the main target of BPA toxicity in impairing the glycemia. They suggest that the BPA exposure can weaken the response of the pancreatic islets to damages. The last observation could represent a broader concept whose consideration should lead to the development of experimental plans better reproducing the multiple exposure conditions.

Dicer is a crucial enzyme for the maturation of miRNAs. Mutations in the Dicer gene are highly associated with Pleuro Pulmonary Blastoma-Family Dysplasia Syndrome (PPB-FDS, OMIM 601200), recently proposed to be renamed Dicer syndrome. Aside from the pulmonary phenotype (blastoma), renal nephroma and thyroid goiter are frequently part of Dicer syndrome. To investigate the renal phenotype, conditional knockout (cKO) mice for Dicer in Pax8 expressing cells were generated. Dicer cKO mice progressively develop a glomerulocystic phenotype coupled with urinary concentration impairment, proteinuria and severe renal failure. Higher cellular turnover of the parietal cells of Bowman's capsule precedes the development of the cysts and the primary cilium progressively disappears with cyst-enlargement. Upregulation of GSK3β precedes the development of the glomerulocystic phenotype. Downregulation of β-catenin in the renal cortex and its cytosolic removal in the cells lining the cysts may be associated with observed accumulation of GSK3β. Alterations of β-catenin regulating pathways could promote cystic degeneration as in other models. Thus, miRNAs are fundamental in preserving renal morphology and function. Alteration of the GSK3β/β-catenin pathway could be a crucial mechanism linking miRNA dysregulation and the development of a glomerulocystic disease.

The eukaryotic GINS complex has an essential role in the initiation and elongation phases of genome duplication. It is composed of four paralogous subunits--Sld5, Psf1, Psf2 and Psf3--which are ubiquitous and evolutionarily conserved in eukaryotic organisms. Here, we report the biochemical characterization of the human GINS complex (hGINS). The four hGINS subunits were coexpressed in Escherichia coli in a highly soluble form and purified as a complex. hGINS was shown to interact directly with the heterodimeric human DNA primase, by using either surface plasmon resonance measurements or by immunoprecipitation experiments carried out with anti-hGINS antibodies. The DNA polymerase alpha-primase synthetic activity was specifically stimulated by hGINS on various primed DNA templates. The significance of these findings is discussed in view of the molecular dynamics at the human replication fork.

We identified the recA gene of the moderately thermophilic bacterium Streptococcus thermophilus and investigated the role of its product in the adaptation to heat shock and nutrient starvation. Expression of recA was required for optimal viability and normal cell morphology upon induction of both stresses. Normal induction of GroEL and ClpL in a recA knock-out mutant suggests that the RecA role in heat shock and nutrient starvation response of S. thermophilus is independent from the intracellular accumulation of these stress-specific chaperones.

Background The molecular mechanisms leading to a fully differentiated thyrocite are still object of intense study even if it is well known that thyroglobulin, thyroperoxidase, NIS and TSHr are the marker genes of thyroid differentiation. It is also well known that Pax8, TTF-1, Foxe1 and Hhex are the thyroid-enriched transcription factors responsible for the expression of the above genes, thus are responsible for the differentiated thyroid phenotype. In particular, the role of Pax8 in the fully developed thyroid gland was studied in depth and it was established that it plays a key role in thyroid development and differentiation. However, to date the bases for the thyroid-enriched expression of this transcription factor have not been unraveled yet. Here, we report the identification and characterization of a functional thyroid-specific enhancer element located far upstream of the Pax8 gene. Results We hypothesized that regulatory cis-acting elements are conserved among mammalian genes. Comparison of a genomic region extending for about 100 kb at the 5'-flanking region of the mouse and human Pax8 gene revealed several conserved regions that were tested for enhancer activity in thyroid and non-thyroid cells. Using this approach we identified one putative thyroid-specific regulatory element located 84.6 kb upstream of the Pax8 transcription start site. The in silico data were verified by promoter-reporter assays in thyroid and non-thyroid cells. Interestingly, the identified far upstream element manifested a very high transcriptional activity in the thyroid cell line PC Cl3, but showed no activity in HeLa cells. In addition, the data here reported indicate that the thyroid-enriched transcription factor TTF-1 is able to bind in vitro and in vivo the Pax8 far upstream element, and is capable to activate transcription from it. Conclusions Results of this study reveal the presence of a thyroid-specific regulatory element in the 5' upstream region of the Pax8 gene. The identification of this regulatory element represents the first step in the investigation of upstream regulatory mechanisms that control Pax8 transcription during thyroid differentiation and are relevant to further studies on Pax8 as a candidate gene for thyroid dysgenesis.

Background Chondroitin sulphate is a complex polysaccharide having important structural and protective functions in animal tissues. Extracted from animals, this compound is used as a human anti-inflammatory drug. Among bacteria, Escherichia coli K4 produces a capsule containing a non-sulphate chondroitin and its development may provide an efficient and cheap fermentative production of the polysaccharide. Results A random N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis was performed on E. coli K4 to isolate mutants showing an increased production of chondroitin. Several mutants were isolated, one of which, here named VZ15, produced about 80% more chondroitin than the wild type E. coli. We found that the mutant has a missense mutation in the codon 313 of kfoC, the gene encoding chondroitin polymerase (K4CP), with a change from arginine to glutamine. A docking analysis to explain the increased productivity of the K4CP enzyme is presented. Conclusion The enhanced chondroitin production by the E. coli K4 mutant reported here shows the validity of the strain improvement strategy for more cost-friendly fermentative processes in the production of this pharmaceutically important but so-far expensive polysaccharide.

We have conditionally inactivated the E-cadherin gene in the thyroid follicular cells of mouse embryo to unravel its role in thyroid development. We used the Cre-loxP system in which the Cre-recombinase was expressed under the control of the tissue-specific thyroglobulin promoter that becomes active at embryonic d 15. At postnatal d 7, thyroid follicle lumens in the knockout mice were about 30% smaller with respect to control mice and had an irregular shape. E-cadherin was almost completely absent in thyrocytes, beta-catenin was significantly reduced, whereas no change in gamma-catenin was detected. alpha-Catenin was also reduced on the cell plasma membrane. Despite the dramatic loss of E-cadherin and beta-catenin, cell-cell junctions were not affected, the distribution of tight junction proteins was unaltered, and no increase of thyroglobulin circulating in the blood was observed. In addition, we found that other members of the cadherin family, the R-cadherin and the Ksp-cadherin, were expressed in thyrocytes and that their membrane distribution was not altered in the E-cadherin conditional knockout mouse. Our results indicate that E-cadherin has a role in the development of the thyroid gland and in the expression of beta-catenin, but it is not essential for the maintenance of follicular cell adhesion.

Mini-chromosome maintenance (MCM) proteins form ring-like hexameric complexes that are commonly believed to act as the replicative DNA helicase at the eukaryotic/archaeal DNA replication fork. Because of their simplified composition with respect to the eukaryotic counterparts, the archaeal MCM complexes represent a good model system to use in analyzing the structural/functional relationships of these important replication factors. In this study the domain organization of the MCM-like protein from Sulfolobus solfataricus (Sso MCM) has been dissected by trypsin partial proteolysis. Three truncated derivatives of Sso MCM corresponding to protease-resistant domains were produced as soluble recombinant proteins and purified: the N-terminal domain (N-ter, residues 1-268); a fragment comprising the AAA+ and C-terminal domains (AAA+-C-ter, residues 269-686); and the C-terminal domain (C-ter, residues 504-686). All of the purified recombinant proteins behaved as monomers in solution as determined by analytical gel filtration chromatography, suggesting that the polypeptide chain integrity is required for stable oligomerization of Sso MCM. However, the AAA+-C-ter derivative, which includes the AAA+ motor domain and retains ATPase activity, was able to form dimers in solution when ATP was present, as analyzed by size exclusion chromatography and glycerol gradient sedimentation analyses. Interestingly, the AAA+-C-ter protein could displace oligonucleotides annealed to M13 single-stranded DNA although with a reduced efficiency in comparison with the full-sized Sso MCM. The implications of these findings for understanding the DNA helicase mechanism of the MCM complex are discussed.

The hyper-thermophilic archaeon Sulfolobus solfataricus possesses two functional DNA polymerases belonging to the B-family (Sso DNA pol B1) and to the Y-family (Sso DNA pol Y1). Sso DNA pol B1 recognizes the presence of uracil and hypoxanthine in the template strand and stalls synthesis 3-4 bases upstream of this lesion ("read-ahead" function). On the other hand, Sso DNA pol Y1 is able to synthesize across these and other lesions on the template strand. Herein we report evidence that Sso DNA pol B1 physically interacts with DNA pol Y1 by surface plasmon resonance measurements and immuno-precipitation experiments. The region of DNA pol B1 responsible for this interaction has been mapped in the central portion of the polypeptide chain (from the amino acid residue 482 to 617), which includes an extended protease hyper-sensitive linker between the N- and C-terminal modules (amino acid residues Asn482-Ala497) and the alpha-helices forming the "fingers" sub-domain (alpha-helices R, R' and S). These results have important implications for understanding the polymerase-switching mechanism on the damaged template strand during genome replication in S. solfataricus.

Ras proteins are highly related GTPases that have key roles in regulating growth, differentiation and tumorigenesis. Gene-targeting experiments have shown that, out of the three mammalian ras genes, only K-ras is essential for normal mouse embryogenesis, and that mice deprived of H-ras and/or N-ras show no major phenotype. We generated mice (HrasKI) in which the K-ras gene had been modified to encode H-Ras protein. HrasKI mice produce undetectable amounts of K-Ras but - in contrast to mice homozygous for a null K-ras allele - they are born at the expected mendelian frequency, indicating that H-Ras can be substituted for K-Ras in embryonic development. However, adult HrasKI mice show dilated cardiomyopathy associated with arterial hypertension. Our results show that K-Ras can be replaced by H-Ras in its essential function in embryogenesis, and indicate that K-Ras has a unique role in cardiovascular homeostasis. © 2005 European Molecular Biology Organization.

The cotG and cotH genes of Bacillus subtilis encode two previously characterized spore coat proteins. The two genes are adjacent on the chromosome and divergently transcribed by sigma(K), a sporulation-specific sigma factor of the RNA polymerase. We report evidence that the cotH promoter maps 812 bp upstream of the beginning of its coding region and that the divergent cotG gene is entirely contained between the promoter and the coding part of cotH. A bioinformatic analysis of all entirely sequenced prokaryotic genomes showed that such chromosomal organization is not common in spore-forming bacilli. Indeed, CotG is present only in B. subtilis, B. amyloliquefaciens, and B. atrophaeus and in two Geobacillus strains. When present, cotG always encodes a modular protein composed of tandem repeats and is always close to but divergently transcribed with respect to cotH. Bioinformatic and phylogenic data suggest that such genomic organizations have a common evolutionary origin and that the modular structure of the extant cotG genes is the outcome of multiple rounds of gene elongation events of an ancestral minigene

Cdc6 proteins play an essential role in the initiation of chromosomal DNA replication in Eukarya. Genes coding for putative homologs of Cdc6 have been also identified in the genomic sequence of Archaea, but the properties of the corresponding proteins have been poorly investigated so far. Herein, we report the biochemical characterization of one of the three putative Cdc6-like factors from the hyperthermophilic crenarchaeon Sulfolobus solfataricus (SsoCdc6-1). SsoCdc6-1 was overproduced in Escherichia coli as a His-tagged protein and purified to homogeneity. Gel filtration and glycerol gradient ultracentrifugation experiments indicated that this protein behaves as a monomer in solution (molecular mass of about 45 kDa). We demonstrated that SsoCdc6-1 binds single- and double-stranded DNA molecules by electrophoretic mobility shift assays. SsoCdc6-1 undergoes autophosphorylation in vitro and possesses a weak ATPase activity, whereas the protein with a mutation in the Walker A motif (Lys-59 --> Ala) is completely unable to hydrolyze ATP and does not autophosphorylate. We found that SsoCdc6-1 strongly inhibits the ATPase and DNA helicase activity of the S. solfataricus MCM protein. These findings provide the first in vitro biochemical evidence of a functional interaction between a MCM complex and a Cdc6 factor and have important implications for the understanding of the Cdc6 biological function.

We report a novel method for preparing samples for laser capture microdissection. The procedure described here permits extraction of intact RNA while preserving morphology, thus being suitable both for identification of specific cells and for analysis of their gene expression. The method is applicable to both mouse embryos and human tumors and may improve the preparation of cDNA libraries from specific cell types without interfering with histological diagnosis. (c)2001 Elsevier Science.

Titf2/foxe1 is a forkhead domain-containing gene expressed in the foregut, in the thyroid, and in the cranial ectoderm of the developing mouse. Titf2 null mice exhibit cleft palate and either a sublingual or completely absent thyroid gland. In humans, mutations of the gene encoding for thyroid transcription factor-2 (TTF-2) result in the Bamforth syndrome, characterized by thyroid agenesis, cleft palate, spiky hair, and choanal atresia. Here, we report a detailed expression pattern of TTF-2 protein during mouse embryogenesis and show its presence in structures where it has not been described yet. At embryonic day (E) 10.5, TTF-2 is expressed in Rathke's pouch, in thyroid, and in the epithelium of the pharyngeal wall and arches, whereas it is absent in the epithelium of the pharyngeal pouches. According to this expression, at E13.5, TTF-2 is present in endoderm derivatives, such as tongue, palate, epiglottis, pharynx, and oesophagus. Later in embryogenesis, we detect TTF-2 in the choanae and whiskers. This pattern of expression helps to define the complex phenotype displayed by human patients. Finally, we show that TTF-2 is a phosphorylated protein. These results help to characterize the domains of TTF-2 expression, from early embryogenesis throughout organogenesis, providing more detail on the potential role of TTF-2 in the development of endoderm and ectoderm derived structures.

We report the characterization of the ccpA gene of Lactobacillus plantarum , coding for catabolite control protein A. The gene is linked to the pepQ gene, encoding a proline peptidase, in the order ccpA-pepQ , with the two genes transcribed in tandem from the same strand as distinct transcriptional units. Two ccpA transcription start sites corresponding to two functional promoters were found, expression from the upstream promoter being autogenously regulated through a catabolite-responsive element ( cre ) sequence overlapping the upstream +1 site. During growth on ribose, the upstream promoter showed maximal expression, while growth on glucose led to transcription from the downstream promoter. In a ccpA mutant strain, the gene was transcribed mainly from the upstream promoter in both repressing and non repressing conditions. Expression of two enzyme activities, β-glucosidase and β-galactosidase, was relieved from carbon catabolite repression in the ccpA mutant strain. In vivo footprinting analysis of the catabolite-controlled bglH gene regulatory region in the ccpA mutant strain showed loss of protection of the cre under repressing conditions.

A newly-identified bglGPT operon of Lactobacillus plantarum was isolated and expressed in Escherichia coli. The sequence analysis of the cloned DNA fragment showed three open reading frames encoding (i) a 237-amino acid protein (BglG), (ii) a 577-amino acid protein (BglP) and (iii) a 486-amino acid protein (BglT). BglG, BglP and BglT were shown to be homologous to the BglG family of transcriptional antiterminators, to permeases of the phosphoenolpyruvate-dependent phosphotransferase system and to β-glucosidases, respectively. Complementation of E. coli mutant strains showed that BglP and BglT are a permease and a β-glucosidase active on the β-glucosides, 5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside and p-nitrophenyl-β-D-glucoside, respectively. BglG was also shown to promote expression of a bglG-lacZ gene fusion in an E. coli bglG- background. A ribonucleic antiterminator sequence, the antiterminator-responsive cis-element and a 'catabolite responsive element', were found downstream of the transcriptional start point. Transcription of the operon was repressed 10-fold in L. plantarum cells grown on glucose as compared to ribose. Copyright (C) 2000 Federation of European Microbiological Societies.

We used a PCR-based method to generate a single base pair mutation in the proB gene of Streptococcus thermophilus, which replaced an aspartic acid with a glycine residue at position 192 of the first proline biosynthetic enzyme gamma-glutamyl kinase. This was the first identified mutation in amino acid biosynthesis in S. thermophilus to our knowledge. The mutation caused an enhanced, feedback-resistant gamma-glutamyl kinase activity and conferred an analogue-resistant phenotype to an Escherichia coli transformant containing the mutated gene.

A newly identified bglH gene coding for a phospho-β-glucosidase of Lactobacillus plantarum was isolated and expressed in Escherichia coli . The sequence analysis of the cloned DNA fragment showed an open reading frame encoding a 480-amino-acid protein with a calculated molecular mass of 53 kDa. The bglH gene was shown to be expressed on a monocistronic transcriptional unit. Its transcription was repressed 10-fold in L. plantarum cells grown on glucose compared to the β-glucoside salicin as a sole carbon source. A catabolite-responsive element (CRE) spanning from −3 to +11 with respect to the transcriptional start point was found, and its functionality was assessed by mutational analysis. In vitro and in vivo DNA binding experiments suggested the occurrence of a DNA-protein complex at the CRE site, which would mediate glucose repression of bglH expression.

The thyroglobulin gene, the substrate for thyroid hormone biosynthesis, is not expressed in the FRT cell line, which, even though it manifests the polarised epithelial phenotype, does not express any of the thyroid functional properties. Two transcription factors, TTF-1 and Pax-8, have been implicated in thyroid specific expression of the thyroglobulin gene. FRT cells contain Pax-8 but they lack TTF-1. In this paper, we show that transfection of TTF-1 expression vectors in FRT cells results in activation of thyroglobulin gene expression. If the expression vector encoded for TTF-1-ER, a fusion gene coding for the entire TTF-1 protein fused to the hormone-binding domain of the steroid receptor, under the control of the RSV promoter, thyroglobulin gene expression was controlled by estrogen. These data provide a direct demonstration that TTF-1 activates the chromosomal thyroglobulin promoter. Since transfection of TTF-1 expression vectors in non-thyroid cell types did not result in thyroglobulin gene expression, it is suggested that Pax-8, in addition, perhaps, to a specific cellular environment, might be required for thyroid specific expression of the thyroglobulin gene. (C) 1997 Elsevier Science B.V.

The Bacillus subtilis BofA protein is involved in regulation of pro-σ K processing in the mother cell during the late stages of sporulation. A computer analysis of the BofA amino acid sequence indicates that it is an integral membrane protein. To determine the membrane topology of the protein, a series of gene fusions of bofA with lacZ or phoA reporter genes in Escherichia coli were analysed. A BofA topological model with two membrane-spanning segments, and with the N- and the C-terminal domains located in the region between the inner and outer membranes surrounding the forespore is presented. The analysis of different modifications of the last five amino acid residues of the BofA protein, obtained by PCR site-directed mutagenesis, suggests a possible role of the C-terminal domain in the regulation of pro-σ K processing.

We report the cloning of a 2-kb PstI-BamHI fragment of Bacillus subtilis DNA carrying an open reading frame of 1398 bp, herein designated orfRM1. This orf was shown to be transcribed only during vegetative growth from a putative sigma A-specific promoter. The deduced amino acid sequence predicted a polypeptide of 51 kDa (466 aa), which shows significant percentage of identity with the Escherichia coli Prc protein. However no Prc-like phenotypes were observed in a B. subtilis orfRM1 deletion-insertion mutant.

Agrobacterium tumefaciens strain C58 was able to utilize carbon from cellobiose and some other beta-D-glucosides as efficiently as from glucose. beta-D-glucoside utilization was partially inducible and the induction was subject to catabolite repression by glucose, independently of the presence of cyclic AMP in the medium. It was also independent of Ti plasmid-encoded functions. beta-D-glucosides were hydrolysed by a single, cytoplasmic and constitutively expressed beta-glucosidase, which was active on non-phosphorylated substrates and insensitive to glucose inhibition.

Transcription of the ilvIH operon was reduced during amino acid starvation of wild-type Escherichia coli. The effect was abolished by a relA mutation and was enhanced by a spoT mutation, thus suggesting a possible negative role of ppGpp accumulation on ilvIH transcription. No effect of amino acid downshift was observed on the synthesis of Irp mRNA, encoding the positive regulator (Lrp) of ilvIH transcription. © 1995.

Acetohydroxy acid synthase (AHAS) and threonine deaminase (TD) activities were found in Streptococcus bovis and shown to be involved in the biosynthesis of the branched chain amino acids isoleucine, leucine and valine. Apparent lack of repression of AHAS synthesis by the end-products and reduced sensitivity of S. bovis growth to analogues of the branched chain amino acids suggested that secretion of isoleucine, leucine and valine in the growth medium may be a consequence of the regulatory features of AHAS. A glycyl-leucine-resistant mutant with reduced TD activity secreted a reduced amount of isoleucine and an increased amount of valine, which might be a result of the reduced rate of synthesis of the isoleucine precursor alpha-ketobutyrate and of a consequent preferential carbon flow through the valine branch of the pathway.

The leucine-responsive regulatory protein (Lrp) is a DNA binding protein that affects, either positively or negatively, the expression of several E. coli genes. The ilvIH operon is positively regulated by Lrp and leucine counteracts this effect reducing 5- to 10-fold the efficiency of ilvIH transcription. An investigation of the mechanism of transcription activation of the ilvIH operon by Lrp indicated that: (i) a stereospecific alignment between the ilvIH promoter and the cis-acting sequence upstream of it is required for activation; (ii) a correct distance between the promoter and the adjacent cis-acting sequence is needed for leucine to counteract the positive role of Lrp; (iii) Lrp fails to activate transcription when the cis-acting region is placed several hundred base pairs upstream of the ilvIH promoter. © 1993.

Lrp, a major regulatory protein in Escherichia coli, controls the expression of numerous operons, including ilvlH. Lrp binds to six sites upstream of ilvlH, and Lrp binding is required for ilvlH expression. We show here that an Lrp‐like protein is also present in Salmonella typhimurium. This protein can bind both E. coli and S. typhimurium ilvlH DNA, as can E. coli Lrp. Methidiumpropyl‐EDTA footprinting studies were performed with purified E. coli Lrp and S. typhimurium ilvlH DNA. Six binding sites were defined, three of them being similar to corresponding sites in E. coli, and three being organized differently. A consensus derived from six S. typhimurium sites is compatible with that derived from a similar analysis of E. coli sequences. Copyright © 1993, Wiley Blackwell. All rights reserved

The cryptic ilvlH locus of Salmonella typhimurium has genetic information for two distinct subunits of acetohydroxy acid synthase III. We show that the ilvH‐encoded subunit is normally translated and the lack of activity is due to early termination of translation within the promoter‐proximal ilvl gene. Analysis of the 5’region of the operon led to identification of the promoter and the amino‐terminal part of ilvl. Expression of this gene in a mutant producing acetohydroxy acid synthase is due to a transversion which creates a UUA (leucine) codon in the place of a UGA (stop) codon present in position 12 of the wild‐type coding region. Copyright © 1991, Wiley Blackwell. All rights reserved

Human T lymphocyte proliferative response induced via CD28 molecule is analyzed. An anti CD28 MoAb, CLB-CD28/1, induces the proliferation of human peripheral blood mononuclear cells in the absence of other stimuli, indicating that CD28 molecule can directly mediate a mitogenic signal in this system. The mitogenic activity of MoAb CLB-CD28/1 on PBMC does not require MoAb interaction with monocyte Fc receptors, since F(ab′)2 fragments from the MoAb are mitogenic to the same extent as whole IgG. Nevertheless, the activity depends on the presence of accessory cells, since purified T lymphocytes require addition of irradiated monocytes and interleukin 2 to proliferate when incubated with MoAb CLB-CD28/1. On the other hand, MoAb CLB-CD28/1 induces response to IL-2 in thymocytes in the absence of accessory cells. Cooperation of MoAb CLB-CD28/1 with three other MoAbs, recognizing CD3, CD5 and HLA Class I antigens, respectively, induces Tac antigen expression and IL-2 responsiveness in purified T lymphocytes. This effect is obtained without cross-linking of the MoAb. It does not rely on a physical association between CD28 and CD3, CD5 or HLA Class I molecules, as demonstrated by co-modulation experiments. These data indicate that expression of IL-2 receptor on T lymphocytes can result from interaction of multiple activation pathways and that some of them, such as those mediated by CD5 and HLA Class I antigens, previously reported to serve as modulatory circuits, can instead act as essential elements in the onset of T lymphocyte proliferation.

A bacteriocin-producing Bacillus cereus strain was isolated. The bacteriocin, here called cerein, was shown to be active specifically against other B. cereus strains and inactive against all other bacterial species tested. Cerein was detected in the culture supernatants of stationary-phase cells, and its appearance was inhibited by induction of sporulation. The bacterial activity of cerein was insensitive to organic solvents and nonproteolytic enzymes, partially stable to heat, and active over a wide range of pH values. Direct detection of antimicrobial activity on sodium dodecyl sulfate-polyacrylamide gel suggested an apparent molecular mass of about 9 kDa.

Endospores of Bacillus subtilis are encased in a protein shell, known as the spore coat, composed of a lamella-like inner layer and an electron-dense outer layer. We report the identification and characterization of a gene, herein called cotH, located at 300 degrees on the B. subtilis genetic map between two divergent cot genes, cotB and cotG. The cotH open reading frame extended for 1,086 bp and corresponded to a polypeptide of 42.8 kDa. Spores of a cotH null mutant were normally heat, lysozyme, and chloroform resistant but were impaired in germination. The mutant spores were also pleiotropically deficient in several coat proteins, including the products of the previously cloned cotB, -C, and -G genes. On the basis of the analysis of a cotE cotH double mutant, we infer that CotH is probably localized in the inner coat and is involved in the assembly of several proteins in the outer layer of the coat.