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PubMed1987

Trehalase of Escherichia coli. Mapping and cloning of its structural gene and identification of the enzyme as a periplasmic protein induced under high osmolarity growth conditions.

Escherichia coli can use the nonreducing disaccharide trehalose as a sole source of carbon and energy. Trehalose transport into the cell is mediated via the phosphotransferase system, and a mutant depleted in the nonspecific proteins enzyme I, HPr, and enzyme IIIGlc of this system was not only unable to grow on glucose or mannitol but also was strongly reduced in its ability to grow on trehalose. A pseudorevertant (PPA69) of such a deletion mutant was isolated that could again grow on glucose but not on mannitol. This revertant could now also use trehalose as a carbon source due to a constitutive galactose permease. PPA69 was subjected to Tn10 insertional mutagenesis, and a mutant (UE5) was isolated that no longer could use trehalose as a carbon source but could still grow on glucose. UE5 lacked a periplasmic trehalase that was present in PPA69. P1-mediated transduction of this Tn10 insertion (treA::Tn10) into a pts+ wild-type strain (MC4100) had no effect on the ability of MC4100 to grow on trehalose but resulted in loss of the periplasmic trehalase activity. The Tn10 insertion was mapped at 26 min on the E. coli linkage map and was 3% cotransducible with trp, in the order treA::Tn10, trp, cys. Trehalase activity in MC4100 was not induced by growth in the presence of trehalose but increased by about 10-fold when 0.6 M sucrose was added to minimal growth medium. Using the in vivo mini-Mu cloning system and growth on trehalose as selection, we cloned the treA gene. A 9-kilobase EcoRI fragment containing treA was subcloned into pBR322. Strains carrying this plasmid (pTRE5) contained about 100-fold higher periplasmic trehalase activity than PPA69 or MC4100. Using polyacrylamide gel electrophoresis, we found a protein of molecular weight 58,000 among the periplasmic proteins of the pTRE5-carrying strain that was absent in UE5. This protein was purified by ammonium sulfate precipitation and DEAE-Sepharose ion-exchange chromatography and contained all the trehalase activity. Minicells containing the treA+ plasmid produced, in addition to three other proteins, the 58,000-dalton protein. Thus, the plasmid carries the structural gene for the periplasmic trehalase and not just a gene involved in the regulation of the enzyme.

Cloning, Molecular↗
PubMed1998

Induced expression of the Legionella pneumophila gene encoding a 20-kilodalton protein during intracellular infection.

The eukaryotic protein synthesis inhibitor cycloheximid has been used by many investigators to selectively radiolabel intracellular bacteria. Although cycloheximide has no direct effect on bacterial gene expression, there are concerns that long-term inhibition of the host cell protein synthesis may have secondary effects on bacterial gene expression. Therefore, prior to further identification and cloning of the macrophage-induced (MI) genes of Legionella pneumophila, the effects of cycloheximide on L. pneumophila-infected U937 cells were evaluated by transmission electron microscopy. Inhibition of protein synthesis of the host cell for 6 h had no major effect on the ultrastructure of the host cell, on the formation of rough endoplasmic reticulum-surrounded replicative phagosome, or on initiation of intracellular bacterial replication. In contrast, by 15 h of cycloheximide treatment, there was profound deterioration in the host cell as well as in the phagosome. To examine protein synthesis by L. pneumophila during the intracellular infection, U937 macrophage-like cells were infected with L. pneumophila, and intracellular bacteria were radiolabeled during a 2-h cycloheximide treatment or following 12 h of cycloheximide treatment. Comparison by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the protein profile of radiolabeled in vitro-grown L. pneumophila to that of intracellularly radiolabeled bacteria showed that 23 proteins were induced in response to the intracellular environment during 2 h of inhibition of host cell protein biosynthesis. Twelve MI proteins of L. pneumophila were artifactually induced due to prolonged inhibition of the host cell protein synthesis. The gene encoding a 20-kDa MI protein was cloned by a reverse genetics technique. Sequence analysis showed that the cloned gene encoded a protein that was 80% similar to the enzyme inorganic pyrophosphatase. Studies of promoter fusion to a promoterless lacZ gene showed that compared to in vitro-grown bacteria, expression of the pyrophosphatase gene (ppa) was induced fourfold throughout the intracellular infection. There was no detectable induction in transcription of the ppa promoter during exposure to stress stimuli in vitro. The ppa gene of L. pneumophila is the first example of a regulated ppa gene which is selectively induced during intracellular infection and which may reflect enhanced capabilities of macromolecular biosynthesis by intracellular L. pneumophila. The data indicate caution in the long-term use of inhibition of host cell protein synthesis to selectively examine gene expression by intracellular bacteria.

Amino Acid Sequence↗
PubMed2000

Interchromosomal insertions. Identification of five cases and a review.

In five families with questionable chromosome rearrangements, we identified an interchromosomal insertion by fluorescent in situ hybridization (FISH). In case 1 with a dir ins (5;11)(p14;q14q24) in three generations, the mentally retarded and microcephalic proband showed a 5p14-->pter deletion. In case 2, a duplication (13)(q21.31--> q31.2) combined with a deletion (11)(q14-->q22) segregated from a reciprocal ins(11;13)(q14q122)(q21.32q31.2), causing a mixed phenotype with psychomotor retardation, caput quadratum, choanal atresia, and pes equinovarus. In case 3, a dir ins (18;5)(q21.3;p13.1p14) was associated with spontaneous abortions, in case 4, the proband with mental retardation, microcephaly, and a heart defect showed a pure trisomy of (12)(q13-->q15), which had segregated from a carrier of an ins (18;12)(p11.3;q13q15). In case 5, a duplication of (10)(q26.3-->q25.2) segregated from an inv ins(5;10)(q15;q26.3q25.2), which was passed on directly from a mother to her son,with mental retardation. In all families the elucidation of the insertional translocation (IT) considerably increased the associated genetic risks of carriers. For the review, we collected data from 81 articles on 87 IT probands on ascertainment, origin, familial transmittance, progeny, and genetic risks of IT carriers. We also discussed the recombinant chromosomes and complex rearrangements associated with ITs, and listed chromosome regions occurring solely as deletions, or solely as duplications, or as both to facilitate genotype/phenotype correlations. We conclude that ITs are rare chromosomal rearrangements with an 1:80,000 incidence, of which nearly 80% were referred because of congenital abnormalities and mental retardation. A maternal origin was seen in 59.5%, a paternal origin in 26.6%, and 13.9% were de novo. No notable difference in fertility between male and female IT carriers was noticed. Bias of ascertainment was excluded in 15 familial cases and led to an estimate of the genetic risks for IT carriers of 32.0-36.0%. The mean size of the inserted regions occurring solely as duplications (n=39) measures 0.96% of the haploid autosomal length (HAL), and of regions solely occurring as deletions (n=14) 0.47% HAL. In the families where both aneusomies occurred, the size of the insertions ranged between 0.22 and 1.21% HAL. Overall, the findings fit with the general idea that a surplus of genetic material is tolerated more easily than a deficiency.

Abnormalities, Multiple↗
PubMed1995

Angiotensin-converting enzyme gene polymorphism is associated with myocardial infarction but not with development of coronary stenosis.

BACKGROUND: Although both genetic and nongenetic factors contribute to the pathogenesis of coronary artery disease, the identification of specific genetic lesions has lagged behind the identification of critical environmental risk factors. A reported association between myocardial infarction (MI) and the insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme (ACE) gene in European men suggests a critical role for this genomic region. However, the generality of this association remains to be determined. It also is not clear at what stage in disease progression the association with the ACE I/D polymorphism becomes important. METHODS AND RESULTS: We evaluated the ACE I/D polymorphism in patients who had undergone coronary angiography (402 men and 295 women) and in 203 representative control subjects. After polymerase chain reaction amplification, genotypes were determined by agarose gel sizing and by hybridization with allele-specific oligonucleotides. After patients were categorized by the degree of coronary artery stenosis and the occurrence of an MI, the distribution of ACE I/D genotypes was evaluated by log linear analysis. Patients were genetically representative of the regional population, and patients with > 60% stenosis of their coronary arteries had the same distribution of ACE I/D genotypes as did patients with < 10% stenosis. However, among patients with stenosis, the occurrence of an MI was significantly associated with the D allele in all patients (odds ratio [OR], 1.59; P = .002) and in men alone (OR, 1.63; P = .006). The lack of significance in women (OR, 1.40; P = .263) is probably due to the fact that only 36 women in the present study had experienced an MI. Furthermore, the association between MI and the ACE I/D polymorphism was independent of blood pressure, smoking habits, and body mass index. CONCLUSIONS: Segregation of the ACE I/D polymorphism is a pervasive genetic risk factor for MI in whites but has no evident effect on the events leading to stenosis of the coronary arteries. This suggests that risk of MI is influenced by two independent processes--atherogenesis that leads to coronary stenosis followed by conversion to MI. The renin-angiotensin system appears to confer significant risk of infarction by influencing the conversion to MI but has no apparent effect on the development of atherostenosis.

Aged↗
PubMed1998

Systematic mutation screening of the pro-opiomelanocortin gene: identification of several genetic variants including three different insertions, one nonsense and two missense point mutations in probands of different weight extremes.

Pro-opiomelanocortin (POMC) is the precursor of melanocortins (adrenocorticotropin: ACTH, beta-endorphin, beta-lipotropin: beta-LPH, corticotropin like intermediate peptide, alpha-, beta- and gamma-melanocyte-stimulating hormone: alpha-, beta- and gamma-MSH) some of which act in the brain to reduce food intake and are potential mediators of leptin action. Recently, three different mutations in the POMC gene (POMC) were identified in two unrelated children that lead to early-onset extreme obesity, adrenal insufficiency, and red hair pigmentation. In the present study we systematically screened the coding region of POMC in 96 extremely obese children and adolescents, 60 healthy underweight individuals and 46 patients with anorexia nervosa (AN) and identified several variants. a) A 9 and an 18 base pair insertion (9bp and 18bp: AGC AGC GGC and AGC AGC GGC AGC AGC GGC, respectively, between codon 73 and 74; 1,2). These in-frame variants lead to the insertion of three or six amino acids (Ser-Ser-Gly; Ser-Ser-Gly-Ser-Ser-Gly) carboxy-terminal to gamma-MSH. Frequencies of the 9bp insertion allele varied between 3 and 5% among the different study groups (Pearson's chi2 P>0.5). b) Both an out-of-frame 6 bp insertion (within codon 176: GGG CCC) leading to the insertion of two amino acids (Arg-Ala) and a premature stop-codon (G-7316-T: Glu-180-Stop) within the gamma-LPH sequence were maternally inherited in an obese female proband. This proband inherited another missense mutation from her father (Glu-188-Gly). c) A missense mutation (G-7016-A; Asp-80-Asn) was observed in a single patient with AN who also harboured the 9bp insertion on a paternally derived haplotype. d) The allelic co-occurence of two silent mutations (C-6982-T and C-7285-T) was detected in two obese subjects. e) Two further silent mutations (C-3832-T; C-7111-G) were detected in an underweight and an obese subject, respectively. We conclude that the POMC gene harbors several different polymorphisms and mutations, none of which can readily be associated with the phenotypes under study.

Adolescent↗
PubMed1985

Construction and identification of recombinant plasmids carrying cDNAs coding for ovine alpha S1-, alpha S2-, beta-, kappa-casein and beta-lactoglobulin. Nucleotide sequence of alpha S1-casein cDNA.

An ovine mammary cDNA library has been constructed from total poly(A)+ RNA isolated from the mammary gland of a lactating ewe, using a classical procedure. Blunt-ended double-stranded cDNAs prepared with reverse transcriptase and nuclease S1 were tailed with dCTP, inserted into the dGMP-tailed PstI site of plasmid pBR322 and cloned in E. coli. Five series of homologous clones representing abundant messenger RNAs (strong hybridization with a single-stranded cDNA probe generated from total poly(A)+ RNA) were selected using each time a different predominant cloned ds-cDNA as probe, then identified by positive hybridization-translation of the cognate mRNA and subsequent immunoprecipitation and electrophoresis of the protein. The lengths of alpha s1-, alpha s2-, beta-, kappa-casein and beta-lactoglobulin mRNAs are in the range of 1.2, 1.1, 1.25, 1.0 and 0.85 kb, respectively, as determined by Northern blotting analysis. Five homologous mRNAs of similar sizes were identified in the porcine species by dot blot hybridization and Northern analyses. The nucleotide sequence of alpha s1-casein mRNA was determined by sequencing, according to Maxam and Gilbert, both a 1080 bp long cloned ds-cDNA and a ss-cDNA (268 nucleotides) generated by 5' extension of a 5' terminal truncated radiolabeled fragment (83 bp) of the relevant ds-cDNA, used as primer for reverse transcription. The 3' non coding region (431 nucleotides, excluding the poly(A) tail) represents 70% of the length of the coding region (618 nucleotides) flanked by a 61 nucleotide 5' region. Comparison of sequences of ovine and bovine, rat and guinea-pig alpha s1-casein mRNAs has revealed a greater homology in the 3' and especially 5' non coding regions. In the reading frame, the conserved regions are essentially those corresponding to the signal peptide and phosphopeptide domains. The derived 206 amino acid sequence of ovine pre-alpha s1-casein differs from that of its bovine counterpart (genetic variant B) by 24 amino acid substitutions and a deletion of 8 amino acid residues occurring in the polypeptide chain of the mature protein. Such a variation (84% homology only) in two phylogenetically closely related species indicates a high rate of evolution of alpha s1-casein.

Amino Acid Sequence↗
PubMed1999

Identification of protease and rpoN-associated genes of uropathogenic Proteus mirabilis by negative selection in a mouse model of ascending urinary tract infection.

Proteus mirabilis, a motile gram-negative bacterium, is a principal cause of urinary tract infections in patients with functional or anatomical abnormalities of the urinary tract or those with urinary catheters in place. Thus far, virulence factors including urease, flagella, haemolysin, various fimbriae, IgA protease and a deaminase have been characterized based on the phenotypic traits conferred by these proteins. In this study, an attempt was made to identify new virulence genes of P. mirabilis that may not have identifiable phenotypes using the recently described technique of signature-tagged mutagenesis. A pool of chromosomal transposon mutants was made through conjugation and kanamycin/tetracycline selection; random insertion was confirmed by Southern blotting of chromosomal DNA isolated from 16 mutants using the aphA gene as a probe. From the total pool, 2.3% (9/397) auxotrophic mutants and 3.5% (14/397) swarming mutants were identified by screening on minimal salts agar and Luria agar plates, respectively. Thirty per cent of the mutants, found to have either no tag or an unamplifiable tag, were removed from the input pool. Then 10(7) c.f.u. from a 96-mutant pool (approximately 10(5) c.f.u. of each mutant) were used as an input pool to transurethrally inoculate seven CBA mice. After 2 d infection, bacteria were recovered from the bladders and kidneys and yielded about 10(5) c.f.u. as an output pool. Dot blot analysis showed that two of the 96 mutants, designated B2 and B5, could not be hybridized by signature tags amplified from the bladder output pool. Interrupted genes from these two mutants were cloned and sequenced. The interrupted gene in B2 predicts a polypeptide of 37.3 kDa that shares amino acid similarity with a putative protease or collagenase precursor. The gene in B5 predicts a polypeptide of 32.6 kDa that is very similar to that encoded by ORF284 of the rpoN operon controlling expression of nitrogen-regulated genes from several bacterial species. The virulence of the two mutants was tested further by co-challenging CBA mice with each mutant and the parental strain. After 1 week of infection, the B2 and B5 mutants were recovered in numbers 100-fold and 1000-fold less than the parental strain, respectively. Using an in vitro assay, it was shown that the B2 mutant had significantly less (P = 0.0001) extracellular protease activity than the wild-type strain. These findings demonstrate that signature-tagged mutagenesis is a viable approach to identify bacterial genes associated with the ability to infect the urinary tract.

Amino Acid Sequence↗
PubMed1998

Identification of macrophage-specific infectivity loci (mil) of Legionella pneumophila that are not required for infectivity of protozoa.

We have recently shown that many mutants of Legionella pneumophila exhibit similar defective phenotypes within both U937 human-derived macrophages and the protozoan host Acanthamoeba (L.-Y. Gao, O. S. Harb, and Y. Abu Kwaik, Infect. Immun. 65:4738-4746, 1997). These observations have suggested that many of the mechanisms utilized by L. pneumophila to parasitize mammalian and protozoan cells are similar, but our data have not excluded the possibility that there are unique mechanisms utilized by L. pneumophila to survive and replicate within macrophages but not protozoa. To examine this possibility, we screened a bank of 5,280 miniTn10::kan transposon insertion mutants of L. pneumophila for potential mutants that exhibited defective phenotypes of cytopathogenicity and intracellular replication within macrophage-like U937 cells but not within Acanthamoeba polyphaga. We identified 32 mutants with various degrees of defects in cytopathogenicity, intracellular survival, and replication within human macrophages, and most of the mutants exhibited wild-type phenotypes within protozoa. Six of the mutants exhibited mild defects in protozoa. The defective loci were designated mil (for macrophage-specific infectivity loci). Based on their intracellular growth defects within macrophages, the mil mutants were grouped into five phenotypic groups. Groups I to III included the mutants that were severely defective in macrophages, while members of the other two groups exhibited a modestly defective phenotype within macrophages. The growth kinetics of many mutants belonging to groups I to III were also examined, and these were shown to have a similar defective phenotype in peripheral blood monocytes and a wild-type phenotype within another protozoan host, Hartmannella vermiformis. Transmission electron microscopy of A. polyphaga infected by three of the mil mutants belonging to groups I and II showed that they were similar to the parent strain in their capacity to recruit the rough endoplasmic reticulum (RER) around the phagosome. In contrast, infection of macrophages showed that the three mutants failed to recruit the RER around the phagosome during early stages of the infection. None of the mil mutants was resistant to NaCl, and the dot or icm NaCl(r) mutants are severely defective within mammalian and protozoan cells. Our data indicated that in addition to differences in mechanisms of uptake of L. pneumophila by macrophages and protozoa, there were also genetic loci required for L. pneumophila to parasitize mammalian but not protozoan cells. We hypothesize that L. pneumophila has evolved as a protozoan parasite in the environment but has acquired loci specific for intracellular replication within macrophages. Alternatively, ecological coevolution with protozoa has allowed L. pneumophila to possess multiple redundant mechanisms to parasitize protozoa and that some of these mechanisms do not function within macrophages.

Acanthamoeba↗
PubMed2006

Identification of four new PITX2 gene mutations in patients with Axenfeld-Rieger syndrome.

PURPOSE: Axenfeld Rieger syndrome (ARS) is an autosomal dominant inherited disorder affecting development of the ocular anterior chamber, abdomen, teeth and facial structures. The PITX2 gene is a major gene encoding a major transcription factor associated with ARS. METHODS: ARS patients were collected from six unrelated families. Patients and their families were ophthalmologically phenotyped and their blood was collected for DNA extraction. We screened the coding region of human PITX2 gene by direct sequencing. The consequences of the mutations described were investigated by generating crystallographic representations of the amino acid changes. In order to better understand the occurrence of glaucoma in ARS patients, we studied the PITX2 gene expression in human embryonic and fetal ocular tissue sections. RESULTS: We identified four novel PITX2 genetic alterations in four unrelated families with ARS. These mutations included two nonsense mutations (E55X and Y121X), an eight nucleotides insertion (1251 ins CGACTCCT) and a substitution (F58L), in familial and sporadic cases of ARS. We also showed for the first time that PITX2 is expressed at early stages of the human embryonic and fetal periocular mesenchyme, as well as at later stages of human development in the fetal ciliary body, ciliary processes, irido corneal angle and corneal endothelium. The human fetal eye PITX2 gene expression pattern reported here for the first time provides a strong basis for explaining the frequent occurrence of glaucoma in patients affected by PITX2 gene mutations. CONCLUSIONS: Two mutations identified affect the homeodomain (E55X and F58L). The E55X nonsense mutation is likely to alter dramatically the DNA-binding capabilities of the PITX2 homeodomain. Furthermore, there is a complete loss of the carboxy-terminal part of the PITX2 protein beyond the site of the mutation. The phenylalanine F58 is known to contribute to the hydrophobic network of the homeodomain. The crystallographic representations of the mutation F58L show that this mutation may change the conformation of the helical core. The F58L mutation is very likely to modify the homeodomain conformation and probably alters the DNA binding properties of PITX2. The other mutations (Y121X and the eight-nucleotide insertion (1251 ins CGA CTC CT) CGA CTC CT, at position 224 in PITX2A) result in partial loss of the C-terminal domain of PITX2. Pitx2 synergistically transactivates the prolactin promoter in the presence of the POU homeodomain protein Pit-1. Pitx2 activity is regulated by its own C-terminal tail. This region contains a highly conserved 14-amino-acid element involved in protein-protein interactions. The C-terminal 39-amino-acid tail represses DNA binding activity and is required for Pitx2 interactions with other transcription factors, for Pitx2-Pit-1 interaction and Pit-1synergism. Pit-1 interaction with the Pitx2 C terminus masks the inhibitory effect and promotes increased DNA binding activity. Thus, the partial or complete loss of the C terminus tail can lead to decreased or absent DNA binding activity and trigger severe ARS phenotypes. Our in situ hybridization results obtained on human embryonic and fetal ocular tissue sections constitute the first molecular histological data providing an explanation for the occurrence of precocious glaucoma in human patients affected by ARS caused by PITX2 mutations. Further structural and biochemical studies are needed for understanding the wide spectrum of clinical phenotypes caused by the increasing number of new PITX2 mutations found in ARS affected patients.

Abdomen↗
PubMed1998

A novel frameshift mutation 840delA and a novel polymorphism D203A in the steroidogenic acute regulatory protein gene in a Japanese patient with congenital lipoid adrenal hyperplasia. Mutations in brief no. 117. Online.

Congenital lipoid adrenal hyperplasia (CLAH) is an autosomalrecessive disorder characterized by impaired production of all steroids including glucocorticoids, mineralocorticoids and sexsteriods. It has recently been reported that mutations in the steriodogenic acute regulatory protein (StAR) gene cause CLAH. We analyzed the StAR gene in a Japanese patient with CLAH. The patient was revealed to be a compound heterozygote bearing a nonsense mutation Q258X, changing codon 258 (CAG) encoding Gln to the stop codon TAG, and a novel framshift mutation 840delA resulting from deletion of one of the three adenosines normally present in codon 238 (AAA), thus leading to a frameshift after codon 237 (Thr) in the StAR gene. The patient was also revealed to be homozygous for a novel missense point mutation D203A, changing codon 203 (GAC) encoding Asp to GCC encoding Ala in the StAR gene. To elucidate the significance of the D203A mutation, we analyzed the StAR gene sequence in twenty normal subjects, and found that all of them were homozygous for the D203A mutation, indicating that the D203A mutation is an innocent polymorphism. In conclusion, we have identified a novel frameshift mutation 840delA which seems to cause 840delA and the first polymorphism D203A in the human StAR gene.

Adrenal Hyperplasia, Congenital↗