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Analysis of the cytosolic hsp70 gene family in Zea mays.

In this study we have analysed the multigene family coding for the cytoplasmic heat shock 70 kDa proteins (hsp70) in Zea mays. Fully degenerate primers were used in a polymerase chain reaction (PCR) to amplify selected regions of the hsp70 genes. Sequence and Southern blot analysis reveals that at least three highly conserved genes exist in maize. In addition, amplification reveals the presence of a conserved intron in all genes examined. Expression analysis shows that the hsp70 genes studied represent members of the inducible and constitutive families. The results obtained may indicate that there are subfamilies of cytoplasmic hsp70 genes expressed in higher plants.

Amino Acid Sequence↗

Sequence analysis reveals that the BTG1 anti-proliferative gene is conserved throughout evolution in its coding and 3' non-coding regions.

The human BTG1 gene (expressing an anti-proliferative function) is an evolutionarily conserved gene homologous to the murine PC3/TIS21 genes. Here, we report the cloning and sequencing of the murine BTG1 coding region and chicken BTG1 cDNA. The putative human and mouse BTG1 proteins are 100% identical; the chicken BTG1 cDNA contains an open reading frame of 170 amino acids with a 91% identity to its human and murine counterparts. The 3'-untranslated region of BTG1 is also highly conserved (82% homology between human and chicken), suggesting that it plays a key role in the regulation of BTG1 expression. These data confirm that BTG1 is phylogenetically highly conserved and that BTG1 and PC3/TIS21 may constitute the first members of a new family of functionally related genes.

Amino Acid Sequence↗

Unravelling angiosperm genome evolution by phylogenetic analysis of chromosomal duplication events.

Conservation of gene order in vertebrates is evident after hundreds of millions of years of divergence, but comparisons of the Arabidopsis thaliana sequence to partial gene orders of other angiosperms (flowering plants) sharing common ancestry approximately 170-235 million years ago yield conflicting results. This difference may be largely due to the propensity of angiosperms to undergo chromosomal duplication ('polyploidization') and subsequent gene loss ('diploidization'); these evolutionary mechanisms have profound consequences for comparative biology. Here we integrate a phylogenetic approach (relating chromosomal duplications to the tree of life) with a genomic approach (mitigating information lost to diploidization) to show that a genome-wide duplication post-dates the divergence of Arabidopsis from most dicots. We also show that an inferred ancestral gene order for Arabidopsis reveals more synteny with other dicots (exemplified by cotton), and that additional, more ancient duplication events affect more distant taxonomic comparisons. By using partial sequence data for many diverse taxa to better relate the evolutionary history of completely sequenced genomes to the tree of life, we foster comparative approaches to the study of genome organization, consequences of polyploidy, and the molecular basis of quantitative traits.

Arabidopsis↗

High allelic diversity in the methyltransferase gene of a phase variable type III restriction-modification system has implications for the fitness of Haemophilus influenzae.

Phase variable restriction-modification (R-M) systems are widespread in Eubacteria. Haemophilus influenzae encodes a phase variable homolog of Type III R-M systems. Sequence analysis of this system in 22 non-typeable H.influenzae isolates revealed a hypervariable region in the central portion of the mod gene whereas the res gene was conserved. Maximum likelihood (ML) analysis indicated that most sites outside this hypervariable region experienced strong negative selection but evidence of positive selection for a few sites in adjacent regions. A phylogenetic analysis of 61 Type III mod genes revealed clustering of these H.influenzae mod alleles with mod genes from pathogenic Neisseriae and, based on sequence analysis, horizontal transfer of the mod-res complex between these species. Neisserial mod alleles also contained a hypervariable region and all mod alleles exhibited variability in the repeat tract. We propose that this hypervariable region encodes the target recognition domain (TRD) of the Mod protein and that variability results in alterations to the recognition sequence of this R-M system. We argue that the high allelic diversity and phase variable nature of this R-M system have arisen due to selective pressures exerted by diversity in bacteriophage populations but also have implications for other fitness attributes of these bacterial species.

Alleles↗

Recombinant bovine uteroglobin at 1.6 A resolution: a preliminary X-ray crystallographic analysis.

Uteroglobin (UG) is a conserved protein which is induced by progesterone and secreted by the epithelia of various mammalian reproductive and respiratory organs. Recombinant bovine uteroglobin (recbUG), consisting of 80 amino acids with a C-terminal His6 tag, was overexpressed in Escherichia coli and purified. The protein was crystallized in two geometric forms, rhomboid and cuneate (wedge-shaped), by the hanging-drop vapour-diffusion method at 295 K. The rhomboid crystals diffracted to a maximum resolution of 1.6 A using synchrotron radiation. These crystals belong to space group P2(1)2(1)2, with unit-cell parameters a = 81.42, b = 82.82, c = 45.26 A, and contain four monomers per asymmetric unit. The cuneate crystals diffracted to 2.35 A resolution using a rotating-anode generator. These crystals belong to space group C222(1), with unit-cell parameters a = 43.39, b = 93.94, c = 77.30 A, and contain two molecules per asymmetric unit.

Animals↗

Does the management of bladder perforation during transurethral resection of superficial bladder tumors predispose to extravesical tumor recurrence?

PURPOSE: We evaluated the impact of a bladder perforation during transurethral resection of superficial bladder tumor on extravesical tumor recurrence and patient prognosis. We also defined potential risk factors for extravesical recurrence prospectively giving emphasis to the management of the perforation. MATERIALS AND METHODS: The medical records of 3,410 patients were reviewed. Parameters recorded included patient age and sex, tumor stage, grade, number, size and location at the time of perforation, the type of bladder perforation (extraperitoneal vs intraperitoneal) and the way the perforation was managed (open surgical repair vs conservative treatment). Logistic regression analysis was used to identify risk factors for extravesical recurrence. Cox regression analysis was used to compare cancer specific survival. RESULTS: A total of 34 cases of bladder perforation were recorded, 4 patients were treated with open surgery and 30 treated conservatively. The 4 patients who underwent open surgery presented with extravesical recurrence after a mean followup of 7.5 months. The remaining 30 patients had no evidence of extravesical recurrence after a mean followup of 60 months (p <0.001). Of the patients with extravesical relapse 3 died of disease. The surgical management of bladder perforation was the best predictor of extravesical recurrence (p <0.001, r = 1.13), followed by an intraperitoneal localization of the perforation (p =0.0003, r = 0.67) and tumor size (p =0.01, r = 0.42). CONCLUSIONS: Surgical repair of a bladder perforation during transurethral resection of bladder tumor increases the risk of extravesical tumor cell recurrence and negatively affects patient prognosis.

Administration, Intravesical↗

NAIM and site-specific functional group modification analysis of RNase P RNA: magnesium dependent structure within the conserved P1-P4 multihelix junction contributes to catalysis.

The tRNA processing endonuclease ribonuclease P contains an essential and highly conserved RNA molecule (RNase P RNA) that is the catalytic subunit of the enzyme. To identify and characterize functional groups involved in RNase P RNA catalysis, we applied self-cleaving ribozyme-substrate conjugates, on the basis of the RNase P RNA from Escherichia coli, in nucleotide analogue interference mapping (NAIM) and site-specific modification experiments. At high monovalent ion concentrations (3 M) that facilitate protein-independent substrate binding, we find that the ribozyme is largely insensitive to analogue substitution and that concentrations of Mg2+ (1.25 mM) well below that necessary for optimal catalytic rate (>100 mM) are required to produce interference effects because of modification of nucleotide bases. An examination of the pH dependence of the reaction rate at 1.25 mM Mg2+ indicates that the increased sensitivity to analogue interference is not due to a change in the rate-limiting step. The nucleotide positions detected by NAIM under these conditions are located exclusively in the catalytic domain, consistent with the proposed global structure of the ribozyme, and predominantly occur within the highly conserved P1-P4 multihelix junction. Several sensitive positions in J3/4 and J2/4 are proximal to a previously identified site of divalent metal ion binding in the P1-P4 element. Kinetic analysis of ribozymes with site-specific N7-deazaadenosine and deazaguanosine modifications in J3/4 was, in general, consistent with the interference results and also permitted the analysis of sites not accessible by NAIM. These results show that, in this region only, modification of the N7 positions of A62, A65, and A66 resulted in measurable effects on reaction rate and modification at each position displayed distinct sensitivities to Mg2+ concentration. These results reveal a restricted subset of individual functional groups within the catalytic domain that are particularly important for substrate cleavage and demonstrate a close association between catalytic function and metal ion-dependent structure in the highly conserved P1-P4 multihelix junction.

Base Sequence↗

The myostatin gene is a downstream target gene of basic helix-loop-helix transcription factor MyoD.

Myostatin is a negative regulator of myogenesis, and inactivation of myostatin leads to heavy muscle growth. Here we have cloned and characterized the bovine myostatin gene promoter. Alignment of the upstream sequences shows that the myostatin promoter is highly conserved during evolution. Sequence analysis of 1.6 kb of the bovine myostatin gene upstream region revealed that it contains 10 E-box motifs (E1 to E10), arranged in three clusters, and a single MEF2 site. Deletion and mutation analysis of the myostatin gene promoter showed that out of three important E boxes (E3, E4, and E6) of the proximal cluster, E6 plays a significant role in the regulation of a reporter gene in C(2)C(12) cells. We also demonstrate by band shift and chromatin immunoprecipitation assay that the E6 E-box motif binds to MyoD in vitro and in vivo. Furthermore, cotransfection experiments indicate that among the myogenic regulatory factors, MyoD preferentially up-regulates myostatin promoter activity. Since MyoD expression varies during the myoblast cell cycle, we analyzed the myostatin promoter activity in synchronized myoblasts and quiescent "reserve" cells. Our results suggest that myostatin promoter activity is relatively higher during the G(1) phase of the cell cycle, when MyoD expression levels are maximal. However, in the reserve cells, which lack MyoD expression, a significant reduction in the myostatin promoter activity is observed. Taken together, these results suggest that the myostatin gene is a downstream target gene of MyoD. Since the myostatin gene is implicated in controlling G(1)-to-S progression of myoblasts, MyoD could be triggering myoblast withdrawal from the cell cycle by regulating myostatin gene expression.

Animals↗

Prone accelerated partial breast irradiation after breast-conserving surgery: preliminary clinical results and dose-volume histogram analysis.

PURPOSE: To report the clinical and dose-volume histogram results of the first 47 patients accrued to a protocol of accelerated partial breast irradiation. Patients were treated in the prone position with three-dimensional conformal radiotherapy after breast-conserving surgery. METHODS AND MATERIALS: Postmenopausal women with Stage T1N0 breast cancer were eligible only after they had first refused to undergo 6 weeks of standard radiotherapy. Planning CT in the prone position was performed on a dedicated table. The postoperative cavity was defined as the clinical target volume, with a 1.5-cm margin added to determine the planning target volume. A total dose of 30 Gy at 6 Gy/fraction was delivered in five fractions within 10 days. RESULTS: The median age of the patients was 67.5 years (range, 51-88 years). The median tumor diameter was 9 mm (range, 1.3-19 mm). In all patients, the prescribed dose encompassed the planning target volume. The mean volume of the ipsilateral breast receiving 100% of the prescription dose was 26% (range, 10-45%), and the mean volume contained within the 50% isodose surface was 47% (range, 23-75%). The lung and heart were spared by treating in the prone position. Acute toxicity was modest, limited mainly to Grade 1-2 erythema. With a median follow-up of 18 months, only Grade 1 late toxicity occurred, and no patient developed local recurrence. CONCLUSION: These data suggest that this approach is well tolerated, with only mild acute side effects and sparing of the heart and lung.

Aged↗

Site-specific mutations of conserved C-terminal residues in aminoglycoside 3'-phosphotransferase II: phenotypic and structural analysis of mutant enzymes.

In order to test the biological importance of amino acids in the C-terminal quarter of aminoglycoside 3'-phosphotransferase II enzyme, seven of the highly conserved residues in this region, His-188, Asp-190, Asp-208, Gly-210, Arg-211, Asp-216 and Asp-220, were changed via site-directed mutagenesis. The phenotype of each mutant was compared to wildtype in terms of antibiotic susceptibilities and enzymatic activities. All of the substitutions either abolished or significantly reduced the resistance of the resulting strains to kanamycin, neomycin, butirosin, ribostamycin, paromomycin, gentamicin A, and G-418. Similarly, enzyme activities in crude extracts were substantially reduced for the mutant strains. Affinity of the enzyme for Mg+2-ATP decreased with His-188, Asp-190, Asp-216 and Asp-220 substitutions as revealed by Km measurements. Secondary structure analysis predicted that substitutions at the conserved residues caused severe conformational distortions at the corresponding regions of the protein.

Amino Acid Sequence↗

The primary structure and expression of the second open reading frame of the polymerase gene of the coronavirus MHV-A59; a highly conserved polymerase is expressed by an efficient ribosomal frameshifting mechanism.

Sequence analysis of a substantial part of the polymerase gene of the murine coronavirus MHV-A59 revealed the 3' end of an open reading frame (ORF1a) overlapping with a large ORF (ORF1b; 2733 amino acids) which covers the 3' half of the polymerase gene. The expression of ORF1b occurs by a ribosomal frameshifting mechanism since the ORF1a/ORF1b overlapping nucleotide sequence is capable of inducing ribosomal frameshifting in vitro as well as in vivo. A stem-loop structure and a pseudoknot are predicted in the nucleotide sequence involved in ribosomal frameshifting. Comparison of the predicted amino acid sequence of MHV ORF1b with the amino acid sequence deduced from the corresponding gene of the avian coronavirus IBV demonstrated that in contrast to the other viral genes this ORF is extremely conserved. Detailed analysis of the predicted amino acid sequence revealed sequence elements which are conserved in many DNA and RNA polymerases.

Amino Acid Sequence↗

Predicting the conformation of proteins from sequences. Progress and future progress.

A new paradigm for predicting the secondary and tertiary structure of functional proteins from sequence data has emerged from detailed models of how natural selection, conservation, and neutral drift, the three fundamental factors in molecular evolution, leave their mark upon protein sequences. Structural information is extracted from a set of aligned homologous sequences via an analysis of patterns of conservation and variation between proteins with quantitatively defined evolutionary relationships. Tertiary structural information is obtained prior to the assignment of secondary structure, where it plays an important role. Throughout, structural predictions are made with the active involvement of a biochemist whose expertise and insight is critical both for making the prediction and in analyzing its successful and unsuccessful parts. Secondary structure predictions are evaluated based on their ability to sustain an effort to model tertiary structure. Several predictions made using the new paradigm can now be compared with those made under the classical paradigm, including a neural network. The results obtained from the new paradigm are clearly superior to those obtained with the classical paradigm, at least within the protein families that were examined.

Amino Acid Sequence↗

Surgical treatment of deep endometriosis and risk of recurrence.

STUDY OBJECTIVE: To evaluate the risk of recurrence of deep endometriosis after conservative surgery. DESIGN: Retrospective analysis (Canadian Task Force classification II-3). SETTING: Tertiary care university hospital. PATIENTS: One hundred fifteen symptomatic patients operated on in our department from 1996 through 2002 with postoperative follow-up of at least 12 months. INTERVENTION: All patients underwent conservative surgery for deep infiltrating endometriosis. MEASUREMENT AND MAIN RESULTS: Risk factors for recurrence of symptoms and clinical findings and for repeated surgery were evaluated by univariate and multivariate analysis. During follow-up, we observed 28 patients with pain recurrence and 15 patients with recurrent clinical findings, and 12 patients required reoperation for deep endometriosis. Recurrence rates of pain and clinical findings during 36 months were 20.5% and 9%, respectively. Multivariate analysis showed that only age was a significant predictor of pain recurrence (OR 0.9, 95% CI 0.81-0.99, p<.05), enhancing the risk in younger patients. Recurrence of clinical signs of deep endometriosis was predicted by obliteration of the pouch of Douglas (OR 1.46, 95% CI 1.16-16.2, p<.05). Reoperation for deep endometriosis was predicted only by the incompleteness of first operation (OR 21.9, 95% CI 3.2-146.5, p<.001). CONCLUSION: Our study indicates that age, obliteration of the pouch of Douglas, and surgical completeness may have a significant influence on the recurrence of the disease.

Actuarial Analysis↗

Analysis of the cellular functions of Escherichia coli operons and their conservation in Bacillus subtilis.

The common assumption of operons as composed of genes that cooperate in a biological process is confirmed here by showing that Escherichia coli operons tend to be composed of genes that belong to the same general class of cellular function. Furthermore, the comparison between the genomic organization of E. coli and that of Bacillus subtilis shows that the genes that are homologous to genes that belong to experimentally characterized E. coli operons tend to cluster in neighboring regions of the genome. This tendency is greater for the subset of E. coli operons whose genes belong to a single functional class. These observations indicate strong evolutionary pressure that, translated into functional constraints, leads to the inclusion of many essential functions in conserved operons and clusters in these two distant species.

Bacillus subtilis↗

Conservation of the prohormone convertase gene family in metazoa: analysis of cDNAs encoding a PC3-like protein from hydra.

A subclass of proteolytic enzymes that correctly cleave precursor proteins at paired basic residues and are structurally related to the bacterial subtilisins has recently been identified. In yeast, a single membrane-bound proteolytic processing enzyme encoded by the kex2 gene has been found, whereas in higher vertebrates cDNAs encoding four distinct enzymes (PC2, PC3, furin, and PACE 4) have been identified. Like kex2, furin (also known as PACE) contains a hydrophobic transmembrane domain, but PC2, PC3, and PACE 4 lack this feature. All five enzymes exhibit striking similarities in their catalytic domains, and this suggests that they have arisen from a common ancestral subtilisin-like gene. We report here the identification of cDNAs encoding a protein that is similar in structure to PC3 from a simple metazoan, Hydra vulgaris (formerly Hydra attenuata). cDNAs encoding two isoforms of this PC3-like enzyme were obtained that differ only in their carboxyl-terminal sequences, probably due to alternative splicing of a common pre-mRNA. Neither form contains a transmembrane domain. Predicted amino acid sequence comparisons revealed that the hydra PC3-like enzyme is 55.4% and 56.7% identical in the catalytic domain to mouse PC3 and human furin, respectively. RNA blot analyses revealed that the PC3-like RNA is expressed predominantly in the hydra body column and not in the head region, although the hydra head contains a high density of nerve cells, which synthesize a variety of neuropeptides. For this reason, we suspect that another proprotein cleavage enzyme isoform may be expressed in head nerve cells. The isolation of a PC3-like cDNA from hydra is consistent with the presence of neuroendocrine cells and indicates that the PC/furin gene family has been well conserved in all metazoa. A simplified nomenclature for the group of mammalian processing proteases is proposed.

Amino Acid Sequence↗

Conservative surgical treatment of pulmonary hydatid disease in children: an analysis of 35 cases.

PURPOSE: Hydatid cyst disease is a major health problem for people who live in endemic countries such as Turkey. The definitive cure for pulmonary hydatidosis is still surgical. This study evaluates our experience of treating pulmonary hydatidosis, focusing on the conservative surgical management of children with this disease. METHODS: We retrospectively analyzed 35 children ranging in age from 3 to 13 years old, treated for pulmonary hydatidosis during a recent 4-year period. RESULTS: The 35 patients had a collective total of 48 cysts. Twenty-seven patients had a single cyst, 7 had unilateral multiple cysts, and 1 had bilateral multiple cysts. Six patients had multiple coexisting liver cysts. The 33 pulmonary cysts were intact and uncomplicated. Of the total 48 cysts, 34 were larger than 5 cm in diameter. Fever, cough, and dyspnea were the most common symptoms and chest radiography gave a correct diagnosis in 96.4% of the patients. Conservative surgical treatment was carried out in 33 of the 35 children (94%). There were few postoperative complications, and no mortality or recurrence in this series. CONCLUSIONS: Parenchyma-saving surgical procedures such as cystotomy and capitonnage are the preferred methods of treatment for pulmonary hydatid disease in childhood. These simple procedures are safe, reliable, and successful. Combined medical treatment can be given but not as an alternative to surgery. Simultaneous surgical procedures for coexisting liver cysts may be preferred because of the lower morbidity rates and hospitalization. Ultrasound or computed tomography scanning should be done to detect coexisting liver cysts in every patient with pulmonary hydatid disease.

Adolescent↗

Analysis of fish ZP1/ZPB homologous genes--evidence for both genome duplication and species-specific amplification models of evolution.

The vertebrate egg envelope is composed of a family of related proteins, the zona pellucida (ZP) proteins, which are characterized by the presence of a conserved zona pellucida domain. Analysis of teleost fish ZP gene sequences has demonstrated that there are no direct orthologues of the mammalian ZPB and ZP1 genes, but that teleost fish contain multiple copies of two classes of genes (ZPXa and ZPXb) that are equally related to ZPB and ZP1. The two classes of genes are further distinguished by expression in liver or ovary, respectively, indicating there was probably an initial duplication event, followed by a switch to hepatic expression of one of the paralogues. This switch was followed in some species by additional amplification of one of the paralogues with the subsequent loss of the other. It is proposed that the expansion of the number of ZPXa and ZPXb genes and the acquisition of dual sites of synthesis are the result of an ancient polyploidization event, followed by additional species-specific gene amplifications.

Animals↗

Hmob3 brain-specific sequence is a part of phylogenetically conserved human MAP1B gene 3'-untranslated region.

Using in vitro and in silico approaches, we demonstrate that the Hmob3 brain-specific sequence is a part of the extended human MAP1B gene 3'UTR encoded by the portion of the exon 7. Previously we had isolated the 1.4 kb Hmob3 clone (Acc. No Y09836) from the human medulla oblongata cDNA library. In the present paper, we report the RT-PCR detection of extended transcripts containing the Hmob3 sequence as well as a fragment of MAP1B mRNA in the human brain mRNA samples. The existence of such transcripts confirms that Hmob3 is transcribed from the MAP1B gene. Thus we report a previously unknown region of the human MAP1B gene which encodes the MAP1B mRNA 3'UTR. The length of the 3'UTR was estimated of 4330 nucleotides. Comparative sequence analysis shows high phylogenetic conservation of MAP1B 3'UTR in mammals. Using Hmob3 as a probe in the Northern blot analysis, we have revealed two different types of transcripts for brain and kidney mRNA samples; in the skeletal muscle sample we detected both transcripts.

Base Sequence↗