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PubMed1989

Sequence conservation in Alu evolution.

A statistical analysis of a set of genomic human Alu elements is based on a published alignment and a recent classification of these sequences. After separation of the Alu sequences into families, the consensus sequences of these families are determined, using the correct weighting of the unidirectional decay of CG-dinucleotides. For, the tenfold greater mutation rate at CG's requires separate consideration of an independent clock at every stage of analysis. The distributions of the substitutions with respect to the new consensus sequences, taking the CG and the non-CG-nucleotide positions separately, lie far closer to the expected distributions than the total diversity. Computer analysis of the folding of RNAs derived from these sequences indicates that RNA secondary structure is conserved among Alu families, suggesting its importance for Alu proliferation and/or function. The folding pattern, further substantiated by a number of compensatory mutations, includes secondary structure domains which are homologous to those observed in 7SL RNA and a defined region of interaction between the two Alu subunits. These results are consistent with a model in which a small number of conserved Alu master genes give rise via retroposition to the numerous copies of Alu pseudogenes, that then diversify by random substitution. The master genes appeared at different periods during evolution giving rise to different families of Alu sequences.

Base Composition↗
PubMed2005

The quantitation of buffering action II. Applications of the formal & general approach.

BACKGROUND: The paradigm of "buffering" originated in acid-base physiology, but was subsequently extended to other fields and is now used for a wide and diverse set of phenomena. In the preceding article, we have presented a formal and general approach to the quantitation of buffering action. Here, we use that buffering concept for a systematic treatment of selected classical and other buffering phenomena. RESULTS: H+ buffering by weak acids and "self-buffering" in pure water represent "conservative buffered systems" whose analysis reveals buffering properties that contrast in important aspects from classical textbook descriptions. The buffering of organ perfusion in the face of variable perfusion pressure (also termed "autoregulation") can be treated in terms of "non-conservative buffered systems", the general form of the concept. For the analysis of cytoplasmic Ca++ concentration transients (also termed "muffling"), we develop a related unit that is able to faithfully reflect the time-dependent quantitative aspect of buffering during the pre-steady state period. Steady-state buffering is shown to represent the limiting case of time-dependent muffling, namely for infinitely long time intervals and infinitely small perturbations. Finally, our buffering concept provides a stringent definition of "buffering" on the level of systems and control theory, resulting in four absolute ratio scales for control performance that are suited to measure disturbance rejection and setpoint tracking, and both their static and dynamic aspects. CONCLUSION: Our concept of buffering provides a powerful mathematical tool for the quantitation of buffering action in all its appearances.

Animals↗
PubMed2002

Identification of a novel human DDX40gene, a new member of the DEAH-box protein family.

The DExH/D-box superfamily of RNA helicases seems to play key roles during RNA metabolism, such as pre-mRNA splicing, ribosome biogenesis, and others. We have cloned a new gene of the DEAH-box protein subgroup, designated DDX40 (DEAD/H-box polypeptide 40 gene). DDX40 contains 3656 nucleotides and codes for a putative 779-amino-acid protein. Sequence analysis of the cDNA product revealed that it contained a DEAH (Asp-Glu-Ala-His) sequence motif and other conserved motifs. The DDX40 protein shared 53% and 43% amino acid identity with human DDX8 and yeast Drh1, respectively, in the conserved region. Northern blot analysis showed that DDX40 was expressed ubiquitously in the eight tissues examined, implying a general physiological function of the protein. We speculate that, like other members of the DExH/D-box superfamily, DDX40 may play roles in pre-mRNA splicing, ribosome biogenesis and other RNA processing functions.

Amino Acid Sequence↗
PubMed1994

A novel nuclear protein binds centromeric alpha satellite DNA.

We have previously reported the identification of a naturally occurring junction between alpha satellite and satellite III DNA on human chromosomes 13, 14 and 21. Direct sequence analysis has shown that the 9 bp alphoid-derived direct repeat sequence (GTGAAAAAG) present at the junction is fully conserved on these chromosomes. A novel protein, pJ alpha, present in HeLa nuclear extracts, binds to the conserved junction sequence. Mutation analysis of the binding site suggests that pJ alpha can recognize one of the two 9 bp repeats and provides some insight into nucleotides that are important for binding. Competition studies support the possibility that this protein binds a significant portion of genomic alpha satellite DNA. Preliminary protein purification experiments have shown that pJ alpha has a molecular weight of 10-15 kDa.

Base Sequence↗
PubMed2002

Survival of GacS/GacA mutants of the biological control bacterium Pseudomonas aureofaciens 30-84 in the wheat rhizosphere.

GacS/GacA comprises a two-component regulatory system that controls the expression of secondary metabolites required for the control of plant diseases in many pseudomonads. High mutation frequencies of gacS and gacA have been observed in liquid culture. We examined whether gacS/gacA mutants could competitively displace the wild-type populations on roots and thus pose a threat to the efficacy of biological control. The survival of a gac mutant alone and in competition with the wild type on roots was examined in the biological control strain Pseudomonas aureofaciens 30-84. In this bacterium, GacS/GacA controls the expression of phenazine antibiotics that are inhibitory to plant pathogenic fungi and enhance the competitive survival of the bacterium. Wheat seedlings were inoculated with strain 30-84, and bacteria were recovered from roots after 21 days in sterile or nonsterile soil to check for the presence of gacS or gacA mutants. Although no mutants were detected in the inoculum, gacS/gacA mutants were recovered from 29 out of 31 roots and comprised up to 36% of the total bacterial populations. Southern hybridization analysis of the recovered gacA mutants did not indicate a conserved mutational mechanism. Replacement series analysis on roots utilizing strain 30-84 and a gacA mutant (30-84.gacA) or a gacS mutant (30-84.A2) demonstrated that although the mutant population partially displaced the wild type in sterile soil, it did not do so in natural soil. In fact, in natural soil final rhizosphere populations of wild-type strain 30-84 starting from mixtures were at least 1.5 times larger than would be predicted from their inoculation ratio and generally were greater than or equal to the population of wild type alone despite lower inoculation rates. These results indicate that although gacS/gacA mutants survive in natural rhizosphere populations, they do not displace wild-type populations. Better survival of wild-type populations in mixtures with mutants suggests that mutants arising de novo or introduced within the inoculum may be beneficial for the survival of wild-type populations in the rhizosphere.

Bacterial Proteins↗
PubMed2001

Comparative sequence analysis of the imprinted Dlk1-Gtl2 locus in three mammalian species reveals highly conserved genomic elements and refines comparison with the Igf2-H19 region.

The Dlk1-Gtl2 domain on mouse chromosome 12 contains reciprocally imprinted genes with the potential to contribute to our understanding of common features involved in imprinting control. We have sequenced this conserved region in the mouse and sheep and included the human sequence in a three species comparison. This analysis resulted in a precise conservation map and identification of highly conserved sequence elements, some of which we have shown previously to be differentially methylated in the mouse. Additionally, this analysis facilitated identification of a CpG-rich tandem repeat array located approximately 13-15 kb upstream of Gtl2. Furthermore, we have identified a third imprinted transcript that overlaps with the last Dlk1 exon in the mouse. This transcript lacks a conserved open reading frame and is probably generated by cleavage of extended Dlk1 transcripts. Because Dlk1 and Gtl2 share many of the imprinting properties of the well-characterized Igf2-H19 domain, it has been proposed that the two regions may be regulated in the same way. Comparative genomic examination of the two domains indicates that although there are similarities, other features are very different, including the location of conserved CTCF-binding sites, and the level of conservation at regulatory regions.

Animals↗
PubMed2000

Identification of functionally important domains in the N-terminal region of telomerase reverse transcriptase.

Telomerase is a ribonucleoprotein reverse transcriptase responsible for the maintenance of one strand of telomere terminal repeats. The key protein subunit of the telomerase complex, known as TERT, possesses reverse transcriptase-like motifs that presumably mediate catalysis. These motifs are located in the C-terminal region of the polypeptide. Hidden Markov model-based sequence analysis revealed in the N-terminal region of all TERTs the presence of four conserved motifs, named GQ, CP, QFP, and T. Point mutation analysis of conserved residues confirmed the functional importance of the GQ motif. In addition, the distinct phenotypes of the GQ mutants suggest that this motif may play at least two distinct functions in telomere maintenance. Deletion analysis indicates that even the most N-terminal nonconserved region of yeast TERT (N region) is required for telomerase function. This N region exhibits a nonspecific nucleic acid binding activity that probably reflects an important physiologic function. Expression studies of various portions of the yeast TERT in Escherichia coli suggest that the N region and the GQ motif together may constitute a stable domain. We propose that all TERTs may have a bipartite organization, with an N-GQ domain connected to the other motifs through a flexible linker.

Amino Acid Sequence↗
PubMed1991

Structural and functional analysis of the origin of conjugal transfer of the broad-host-range IncW plasmid R388 and comparison with the related IncN plasmid R46.

We cloned and sequenced a 402 bp DNA segment containing the origin of conjugal transfer (oriT) of the IncW plasmid R388. Progressive deletions from each end of the sequence were assayed for oriT activity. Stepwise reductions in mobilization frequencies, representing the loss of functional elements, correlated with deletion of structural motifs in the sequence. A sequence of 330 bp of oriT was sufficient for efficient mobilization. The first 86 bp of the sequence contains five tandemly repeated DNA sequences of 11 bp, followed by a 10 bp perfect inverted repeat. Deletion of the first 95 bp reduced the frequency of transfer by a hundred-fold. The sequence between bp 183 and 218 was necessary and sufficient for low frequency mobilization and, thus, it was assumed to contain the nick site. This basis core was cloned as a 60 bp segment (from bp 176-236) that could be mobilized at low frequency. It includes two inverted repeats and a perfect integration host factor (IHF) consensus binding site. A third functionally important segment in oriT was located between bp 260 and 330. The DNA sequence of the oriT of R388 could be aligned with that of the broad-host-range IncN plasmid R46. Moreover, the relative positions of the three inverted repeats are also conserved. Overall sequence similarity was 52%, but was significantly higher in particular regions, which coincided with the functionally important segments mapped by deletion analysis. Conservation of these segments provided independent support for their essential role in oriT function.

Base Sequence↗
PubMed2001

A cDNA for nuclear-encoded chloroplast translational initiation factor 2 from a higher plant is able to complement an infB Escherichia coli null mutant.

Formation of the initiation translation complex containing the three initiation factors, IF1, IF2, and IF3, tRNA(fMet), and GTP constitutes the earliest event in the protein synthesis. IF2, a GTP-binding protein, is the principal factor involved in selecting and binding fMet-tRNA(fMet) to the 30 S ribosomal subunit. Although some chloroplast initiation translational factors have been identified and purified from algae, none of these factors have been characterized from plants. In this work, we report the molecular characterization of a nuclear-encoded chloroplastic IF2 gene from common bean (PvIF2cp). We show that the PvIF2cp gene encodes a protein containing a chloroplast translocation signal peptide, able to target a green fluorescent protein fusion protein to chloroplasts. A high accumulation of PvIF2cp transcript was found in photosynthetic tissues, whereas low mRNA levels were detected in etiolated plants and in nonphotosynthetic organs. Additional data indicate that the PvIF2cp transcript accumulation is modulated by light. The PvIF2cp gene encodes a functional factor, since the PvIF2cp conserved region, containing the G-domain and the C-terminal end, complements an Escherichia coli infB null mutation. Phylogenetic analysis using the PvIF2cp conserved region suggests that the PvIF2cp gene originated via endosymbiotic gene transfer to the nucleus and that it may be a useful marker for phylogeny reconstruction.

Active Transport, Cell Nucleus↗
PubMed2001

Conserved key amino acid positions (CKAAPs) derived from the analysis of common substructures in proteins.

An all-against-all protein structure comparison using the Combinatorial Extension (CE) algorithm applied to a representative set of PDB structures revealed a gallery of common substructures in proteins (http://cl.sdsc.edu/ce.html). These substructures represent commonly identified folds, domains, or components thereof. Most of the subsequences forming these similar substructures have no significant sequence similarity. We present a method to identify conserved amino acid positions and residue-dependent property clusters within these subsequences starting with structure alignments. Each of the subsequences is aligned to its homologues in SWALL, a nonredundant protein sequence database. The most similar sequences are purged into a common frequency matrix, and weighted homologues of each one of the subsequences are used in scoring for conserved key amino acid positions (CKAAPs). We have set the top 20% of the high-scoring positions in each substructure to be CKAAPs. It is hypothesized that CKAAPs may be responsible for the common folding patterns in either a local or global view of the protein-folding pathway. Where a significant number of structures exist, CKAAPs have also been identified in structure alignments of complete polypeptide chains from the same protein family or superfamily. Evidence to support the presence of CKAAPs comes from other computational approaches and experimental studies of mutation and protein-folding experiments, notably the Paracelsus challenge. Finally, the structural environment of CKAAPs versus non-CKAAPs is examined for solvent accessibility, hydrogen bonding, and secondary structure. The identification of CKAAPs has important implications for protein engineering, fold recognition, modeling, and structure prediction studies and is dependent on the availability of structures and an accurate structure alignment methodology. Proteins 2001;42:148-163.

Algorithms↗
PubMed1993

Mutational analysis of the human DNA polymerase alpha. The most conserved region in alpha-like DNA polymerases is involved in metal-specific catalysis.

Five site-directed mutations were introduced at the most conserved amino acids in region I (YGDTDS) of the human DNA polymerase alpha catalytic subunit. Mutant proteins were expressed in the baculovirus system by an improved method and purified by a rapid one-step purification in high yield and high specific activity. The Asp1004 to Asn mutation produced a protein with no detectable polymerase activity while other mutations gave activities from 1 to 20% of the wild type polymerase activity. Steady state kinetic analysis of the active mutants indicates that none of the mutations caused a change in Km(dNTP) or KD(DNA), but all active mutants showed a decrease in kcat. Thus, the effect of these conserved mutations is manifest in altered rates of catalysis. Two mutations, Asp1002 to Asn and Thr1003 to Ser, caused the enzyme to utilize Mn2+ more effectively in catalysis than Mg2+, suggesting that these amino acids are involved in metal binding. Rates of catalysis by the D1002N and T1003S mutants, as well as Y1000F mutant were improved 80-, 30-, and 70-fold, respectively, on homopolymer templates when Mn2+ replaced Mg2+ as the activator metal. The results from these mutational studies suggest that this highly conserved region binds the metal which is essential for catalysis. The Asp1002 may participate directly in chelating the metal. Results from the T1003S mutant suggest that the beta-methyl group of the threonine side chain might be locked in a hydrophobic pocket preventing free rotation around the C alpha-C beta bond, thus positioning the Thr1003 hydroxyl group to form a crucial bond with the metal ion. In addition, D1002N and T1003S displayed a 20-fold resistance to aphidicolin compared to the wild type polymerase alpha, and all of the active mutants displayed altered sensitivity to butylphenyl-dGTP. Models of the involvement of region I in catalysis and aphidicolin interaction are proposed. The mutational studies presented in this report will serve as a prototype for the functional role of region I in catalysis for all alpha-like DNA polymerases.

Amino Acid Sequence↗
PubMed2005

Conservation and development in Amazonian extractive reserves: the case of Alto Juruá.

Extractive reserves constitute an innovative approach to match conservation and development objectives, which were originally envisaged as part of a land struggle by forest dwellers in Brazil. In spite of the idea's popularity and the attempts to apply the concept to different tropical regions, there has been little analysis of the combined conservation and development performance of extractive reserve programs. We present a detailed analysis of deforestation and demographic and socioeconomic changes in Alto Juruá, the first extractive reserve created in Brazil in 1990. Forest cover has remained fairly stable. Population has declined slightly, with some internal displacements. The cash economy base has shifted from the original rubber production to a diversified portfolio of agriculture and livestock, and there has been a dramatic rise in nonagrarian income. We conclude that the Reserve represents a very dynamic setting with positive conservation and development outcomes during its first decade.

Agriculture↗
PubMed2004

Structure-function analysis of the human sialyltransferase ST3Gal I: role of n-glycosylation and a novel conserved sialylmotif.

All eukaryotic sialyltransferases have in common the presence in their catalytic domain of several conserved peptide regions (sialylmotifs L, S, and VS). Functional analysis of sialylmotifs L and S previously demonstrated their involvement in the binding of donor and acceptor substrates. The region comprised between the sialylmotifs S and VS contains a stretch of four highly conserved residues, with the following consensus sequence (H/y)Y(Y/F/W/h)(E/D/q/g). (Capital letters and lowercase letters indicate a strong or low occurrence of the amino acid, respectively.) The functional importance of these residues and of the conserved residues of motif VS (HX(4)E) was assessed using as a template the human ST3Gal I. Mutational analysis showed that residues His(299) and Tyr(300) of the new motif, and His(316) of the VS motif, are essential for activity since their substitution by alanine yielded inactive enzymes. Our results suggest that the invariant Tyr residue (Tyr(300)) plays an important conformational role mainly attributable to the aromatic ring. In contrast, the mutants W301F, E302Q, and E321Q retained significant enzyme activity (25-80% of the wild type). Kinetic analyses and CDP binding assays showed that none of the mutants tested had any significant effect in nucleotide donor binding. Instead the mutant proteins were affected in their binding to the acceptor and/or demonstrated lower catalytic efficiency. Although the human ST3Gal I has four N-glycan attachment sites in its catalytic domain that are potentially glycosylated, none of them was shown to be necessary for enzyme activity. However, N-glycosylation appears to contribute to the proper folding and trafficking of the enzyme.

Amino Acid Sequence↗
PubMed2002

Lineage specificity of the evolutionary dynamics of the mtDNA D-loop region in rodents.

This paper reports an intraorder study on the D-loop-containing region of the mitochondrial DNA in rodents. A complete multialignment of this region is not feasible with the exception of some conserved regions. The comparative analysis of 25 complete rodent sequences from 23 species plus one lagomorph has revealed that only the central domain (CD), a conserved region of about 80 bp in the extended termination-associated sequences (ETAS) domain, adjacent to the CD, the ETAS1, and conserved sequence block (CSB) 1 blocks are present in all rodent species, whereas the presence of CSB2 and CSB3 is erratic within the order. We have also found a conserved region of 90 bp located between tRNAPro and ETAS1 present in fat dormouse, squirrel, guinea pig, and rabbit. Repeated sequences are present in both the ETAS and the CSB domain, but the repeats differ in length, copy number, and base composition in different species. The potential use of the D-loop for evolutionary studies has been investigated; the presence/absence of conserved blocks and/or repeated sequences cannot be used as a reliable phylogenetic marker, since in some cases they may be shared by distantly related organisms but not by close ones, while in other ones a relationship between tree topology and presence/absence of such motifs is observed. Better results can be obtained by the use of the CD, which, however, due to its reduced size, when used for tracing a phylogenetic tree, shows some nodes with low statistical support.

Animals↗
PubMed1997

Sequence analysis of the terminal protein precursor coding regions from bovine adenovirus serotypes 2 and 3.

As part of our efforts to develop bovine adenoviruses into a vector system, we sequenced the region predicted to encode the terminal protein precursor of either bovine adenoviruses type 2 and bovine adenoviruses type 3. We examined the regions containing the terminal protein precursors and showed that the bovine adenovirus early region 2B had an identical organization to all adenoviruses so far examined. The bovine adenovirus terminal protein precursors and those of other adenoviruses were also compared in a sequence alignment analysis and several highly conserved structural domains were identified. Finally, we showed how the various terminal protein precursors were related in a sequence pair similarity analysis and concluded that the terminal protein precursors of bovine adenoviruses types 2 and 3 are significantly different from each other at the protein sequence level.

Amino Acid Sequence↗
PubMed1999

Analysis of the mouse MAP1B gene identifies a highly conserved 4.3 kb 3' untranslated region and provides evidence against the proposed structure of DBI-1 cDNA.

We determined the previously unknown 3' end of MAP1B mRNA. We found an unusually long and highly conserved 3' untranslated region (3'UTR) of 4.3 kb and detected a polymorphism in the 3' flanking region probably due to the integration of an endogenous retroviral MuERV-L element. Furthermore, we found that MAP1B 3'UTR overlapped with the 5' end of the cDNA encoding DBI-1. However, further analysis suggested that the published structure of DBI-1 cDNA is most likely the result of fortuitous joining of unrelated cDNA fragments during cloning.

Animals↗
PubMed2004

The active site of the ribosome is composed of two layers of conserved nucleotides with distinct roles in peptide bond formation and peptide release.

Peptide bond formation and peptide release are catalyzed in the active site of the large subunit of the ribosome where universally conserved nucleotides surround the CCA ends of the peptidyl- and aminoacyl-tRNA substrates. Here, we describe the use of an affinity-tagging system for the purification of mutant ribosomes and analysis of four universally conserved nucleotides in the innermost layer of the active site: A2451, U2506, U2585, and A2602. While pre-steady-state kinetic analysis of the peptidyl transferase activity of the mutant ribosomes reveals substantially reduced rates of peptide bond formation using the minimal substrate puromycin, their rates of peptide bond formation are unaffected when the substrates are intact aminoacyl-tRNAs. These mutant ribosomes do, however, display substantial defects in peptide release. These results reveal a view of the catalytic center in which an inner shell of conserved nucleotides is pivotal for peptide release, while an outer shell is responsible for promoting peptide bond formation.

Base Sequence↗
PubMed1992

Microinvasive ductal carcinoma of the breast treated with breast-conserving surgery and definitive irradiation.

An analysis was performed of 39 consecutive women with microinvasive ductal carcinoma of the breast treated with breast-conserving surgery and definitive irradiation during the period 1977 to 1988. Microinvasive ductal carcinoma was defined as predominantly intraductal carcinoma with microscopic or early invasion. Surgical treatment of the primary tumor included excisional biopsy or wide resection. Axillary lymph node staging showed that 37 patients were pathologically node negative and two patients were pathologically node positive, each with only one positive lymph node. The median follow-up was 55 months (mean = 65 months; range = 25-135 months). The 5-year actuarial rate of overall and cause-specific survival were both 97%. The 5-year actuarial rate of freedom from distant metastases was 93%. Nine patients developed a recurrence in the breast; eight of the nine patients had isolated local only first failures, and one of the nine patients had a local recurrence simultaneously with distant metastases. The median time to local failure was 42 months (mean = 53 months; range = 20-116 months). Of the eight patients with local only first failure, seven patients have been salvaged with further treatment and remain free of disease at the time of last follow-up, and one patient has died of subsequent distant metastatic disease. Median follow-up after salvage treatment was 29 months (mean = 27 months; range = 0-54 months). Comparison of the patients with microinvasive ductal carcinoma with two control groups of intraductal carcinoma and invasive ductal carcinoma was performed. Although the rate of local failure was significantly higher for patients with microinvasive ductal carcinoma as compared to the two control groups, the rates of survival and freedom from distant metastases for patients with microinvasive ductal carcinoma were intermediate to the two control groups. Because of the high rates of survival and freedom from distant metastases and because of the ability to salvage patients with local recurrence, breast-conserving surgery and definitive irradiation should continue to be considered as an alternative to mastectomy for appropriately selected and staged patients with microinvasive ductal carcinoma of the breast.

Adult↗