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PubMed1981

Human prolactin. cDNA structural analysis and evolutionary comparisons.

Prolactin (Prl), growth hormone, and chorionic sommatomammotropin form a set (the "Prl set") of hormones which is thought to have evolved from a common ancestral gene. This assumption is based on several lines of evidence: overlap in their biological and immunological properties, similarities in their amino acid sequences, and homologies in the nucleic acid sequences of their structural genes. In the current study we report the cloning, amplification in bacteria, and sequence analysis of DNA complementary to Prl mRNA isolated from human pituitary Prl-secreting adenomas. The cloned DNA contains 914 bases, which includes the entire coding sequence of human prePrl as well as portions of the 5- and 3'-untranslated regions of the mRNA. The amino acid sequence predicted by our data differs from a previously reported amino acid sequence in 8 positions. With the results of this study we can now compare in one species the nucleotide sequences of the structural gene coding for each of the hormones of the Prl set. The sequence divergence at replacement sites is used to establish an evolutionary clock for the Prl set of genes. Using this clock, we postulate that the chromosomal segregation of human Prl and human growth hormone occurred about 392 million years ago and that growth hormone and chorionic sommatomammotropin underwent an intrachromosomal recombination within the last 10 million years.

Adenoma↗

PubMed1983

Sequence of rat intestinal vitamin D-dependent calcium-binding protein derived from a cDNA clone. Evolutionary implications.

We have recently reported molecular cloning of the cDNA synthesized from rat duodenal mRNA-encoding intestinal calcium-binding protein (ICaBP), a vitamin D3-induced protein (Desplan, C., Thomasset, M., and Moukhtar, M. S. (1983) J. Biol. Chem. 258, 2762-2765). Nucleotide sequence analysis of the longest cDNA insert (375 base pairs) permitted the assignment of 207 nucleotides of the coding region and 104 nucleotides of the entire 3'-noncoding region of the mRNA. Although the derived amino acid sequence for rat ICaBP differed from the bovine and porcine sequences by 16 and 14 residues, respectively, all the residues of each calcium-binding site met the proposed requirements of the "EF hand" theory. In contrast, several differences found in the linker regions might explain the absence of cross-immunoreactivity between rat and porcine ICaBPs. Analysis of nucleotide sequence homologies between the coding and noncoding regions showed that the region coding for the two calcium-binding sites (I and II) was immediately followed in the noncoding region by a sequence very similar to the sequence coding for site I. This suggests that rat ICaBP mRNA contains the remains of an untranslated calcium-binding site III-like structure and that low Mr ICaBP could result in early termination of the translation of a larger molecule containing four sites.

Amino Acid Sequence↗

PubMed1999

Linear mitochondrial plasmids of F. oxysporum are novel, telomere-like retroelements.

Diverse types of linear RNA and DNA autonomously replicating genetic elements exist in prokaryotic and eukaryotic hosts, yet linear elements that replicate by reverse transcription have not been identified. Here, we report the sequence and organization of two linear mitochondrial plasmids of the fungal plant pathogen F. oxysporum and the characterization of a plasmid-associated reverse transcriptase activity. Plasmids pFOXC2 and pFOXC3 are 1.9 kb in length and have a "clothespin" genomic structure, which includes a terminal hairpin and a telomere-like iteration of a 5 bp sequence at the other terminus. The retroplasmid replication cycle involves novel strategies for copying terminal sequences, which may provide clues concerning the origin of telomerase as well as the evolution of linear DNAs.

Base Sequence↗

PubMed1997

The HsdR subunit of R.EcoR124II: cloning and over-expression of the gene and unexpected properties of the subunit.

Type I restriction endonucleases are composed of three subunits, HsdR, HsdM and HsdS. The HsdR subunit is absolutely required for restriction activity; while an independent methylase is composed of HsdM and HsdS subunits. DNA cleavage is associated with a powerful ATPase activity during which DNA is translocated by the enzyme prior to cleavage. The presence of a Walker type I ATP-binding site within the HsdR subunit suggested that the subunit may be capable of independent enzymatic activity. Therefore, we have, for the first time, cloned and over-expressed the hsdRgene of the type IC restriction endonuclease EcoR124II. The purified HsdR subunit was found to be a soluble monomeric protein capable of DNA- and Mg2+-dependent ATP hydrolysis. The subunit was found to have a weak nuclease activity both in vivo and in vitro, and to bind plasmid DNA; although was not capable of binding a DNA oligoduplex. We were also able to reconstitute the fully active endonuclease from purified M. EcoR124I and HsdR. This is the first clear demonstration that the HsdR subunit of a type I restriction endonuclease is capable of independent enzyme activity, and suggests a mechanism for the evolution of the endonuclease from the independent methylase.

Adenosine Triphosphatases↗

PubMed1980

The cDNA for the beta-subunit of human chorionic gonadotropin suggests evolution of a gene by readthrough into the 3'-untranslated region.

A 579-base pair approximately full-length cDNA which codes for the 145-amino acid long beta-subunit of human chorionic gonadotropin (HCG) has been cloned in the plasmid vector pBR322 and its complete nucleotide sequence determined. A hydrophobic presequence of 20 amino acids can be identified from the nucleotide sequence. The amino acid sequence of the beta-subunit is known to be related to those of the beta-subunits of the other glycoprotein hormones LH, FSH and TSH, but the beta-subunit of HCG is unique in that it contains a C-terminal extension of about 30 amino acids which has no homologous counterpart in the other three hormones. Analysis of the beta HCG cDNA nucleotide sequence suggests that this extension may have arisen by the loss of the termination codon of an ancestral beta-like gene so that most of what was previously the 3'-untranslated region now codes for protein. The beta-subunit of HCG terminates with the codon UAA located 16 bases before the poly(A) in the sequence AAUAAA. This sequence is believed to be a recognition signal involved in either polyadenylation or processing and therefore has a dual role in this gene, serving both a coding and a regulatory function.

Base Sequence↗

PubMed2003

Products transcribed from rearranged rrn genes of Escherichia coli can assemble to form functional ribosomes.

To examine the flexibility of rRNA operons with respect to fundamental organization, transcription, processing, and assembly of ribosomes, operon variations were introduced by a plasmid into an Escherichia coli strain that has deletions of all chromosomal copies of rRNA genes. In the reconstructed operons, a Salmonella intervening sequence (IVS) from 23S helix 45 was introduced into the E. coli 23S gene at the same position. Three different constructs of the E. coli 16S gene were then placed wholly within the IVS sequence, and the 16S gene was deleted from its normal position. The resulting plasmids thus had the normal operon promoters and the leader region followed by the 5' one-third of the 23S gene, the entire 16S gene within the IVS, the last two-thirds of the 23S gene, and the normal end of the operon. The three constructs differed in the amount of 16S leader and spacer regions they contained. Only two of the three constructs, those with redundant leader and spacer antiterminator signals, resulted in viable cultures of the rrn deletion strain. Electron micrographs of the variant operon suggest that the 23S rRNA is made in two separate parts which then must form subassemblies before assembling into a functional 50S subunit. Cells containing only the reshuffled genes were debilitated in their growth properties and ribosome contents. The fact that such out of the ordinary manipulation of rRNA sequences in E. coli is possible paves the way for detailed analysis of ribosome assembly and evolution.

DNA, Ribosomal Spacer↗

PubMed1990

Sequence analysis and evolutionary perspectives of ROB-1 beta-lactamase.

The nucleotide sequence of the ROB-1 beta-lactamase gene from Haemophilus influenzae plasmid RRob was determined. The structural gene encodes a polypeptide of 305 amino acids, with an estimated molecular mass of 30,424 for the mature form of the protein. The ROB-1 gene showed low homologies with other beta-lactamases at the nucleic acid level. By using two statistical computer methods, assessment of the extent of similarity between ROB-1 and other known beta-lactamase amino acid sequences suggested that ROB-1 is a class A enzyme. Alignment of class A beta-lactamases with ROB-1 identified conserved residues. The use of a mutation matrix for detecting distance relationships indicated that ROB-1 has higher values and homologies with beta-lactamases of gram-positive bacteria, giving insight into its ancestry and divergence.

Amino Acid Sequence↗

PubMed2005

New approaches to the analysis of palindromic sequences from the human genome: evolution and polymorphism of an intronic site at the NF1 locus.

The nature of any long palindrome that might exist in the human genome is obscured by the instability of such sequences once cloned in Escherichia coli. We describe and validate a practical alternative to the analysis of naturally-occurring palindromes based upon cloning and propagation in Saccharomyces cerevisiae. With this approach we have investigated an intronic sequence in the human Neurofibromatosis 1 (NF1) locus that is represented by multiple conflicting versions in GenBank. We find that the site is highly polymorphic, exhibiting different degrees of palindromy in different individuals. A side-by-side comparison of the same plasmids in E.coli versus. S.cerevisiae demonstrated that the more palindromic alleles were inevitably corrupted upon cloning in E.coli, but could be propagated intact in yeast. The high quality sequence obtained from the yeast-based approach provides insight into the various mechanisms that destabilize a palindrome in E.coli, yeast and humans, into the diversification of a highly polymorphic site within the NF1 locus during primate evolution, and into the association between palindromy and chromosomal translocation.

Alleles↗

PubMed1993

A comparison of the multiple alleles of xylS carried by TOL plasmids pWW53 and pDK1 and its implications for their evolutionary relationship.

Both of the independently isolated TOL plasmids pWW53 and pDK1 contain multiple regions homologous to the xylS regulatory gene of the archetypal TOL plasmid pWW0. The three homologues on pWW53 vary in the extent of their homology to xylSpWW0, xylS1pWW53 is 99% identical to xylSpWW0 and is located relative to the single copy of xylRpWW53 in exactly the same way as xylS and xylR on pWW0. The DNA sequence of xylS3pWW53 is 87% identical to the xylSpWW0 sequence within the coding region but the non-coding DNA upstream is not homologous. There is a frame-shift change at the end of the coding region which causes the C terminus of XylS3pWW53 to be extended by an additional 10 amino acids relative to XylSpWW0. xylS2pWW53 is anomalous and appears to encode a truncated pseudogene lacking the first 525 bases found in the other xylS genes. Evidence is presented to show that both xylS1pWW53 and xylS3pWW53 act as regulators of meta pathway operons. Plasmid pDK1 carries two homologues of xylS. xylS1pDK1 is functional and is a hybrid gene: its 5' end and the upstream sequences are highly homologous to both xylS1pWW53 and xylSpWW0, whereas its 3' end is identical to xylS3pWW53. The sequence of xylS2pDK1 is identical to that of the anomalous truncated xylS2pWW53. Comparison of the organization and the restriction maps of the xyl catabolic operons on pDK1 and pWW53, together with the nucleotide sequences presented here, indicates that the catabolic DNA on pDK1 has derived from a replicon on which the xyl genes are organized similarly to pWW53 and that a genetic rearrangement has taken place involving a reciprocal recombination internal to two of its xylS homologues.

Alleles↗

PubMed1975

Transposition of R factor genes to bacteriophage lambda.

Transpositions of segments of R factor (antibiotic resistance plasmids) to bacteriophage lambda have been selected and characterized. Cells of Escherichia coli harboring R factors that determine kanamycin resistance were infected with phage lambda, and lambdakan transducing lines were obtained. Each of the three examined is unusual when compared to lambda transducing phages containing E. coli chromosomal genes: the kan insertions (a) occur at several sites, each well removed from the integration region POP', (b) are not associated with deletion of lambda phage DNA, and (c) are separable from the lambda genome during transduction or during lytic growth. Two insertions from the same R factor contain 1.5 kilobase sequences repeated in inverted order. The properties of the lambdakan phage suggest that R factors contain systems capable of mediating genetic exchange in the absence of extensive DNA homology. It is suggested that such systems of exchange may have played important roles in R factor evolution.

Chromosome Mapping↗

PubMed1983

Molecular cloning of human gastrin cDNA: evidence for evolution of gastrin by gene duplication.

An oligo(dT)-primed cDNA copy of the mRNA coding for the human gastrin precursor was constructed from poly(A)-containing RNA from a human pancreatic, gastrin-producing tumor (a gastrinoma). The cDNA was inserted into the Pst I endonuclease site of plasmid pBR322 by the use of the poly(dC) and poly(dG) tailing procedure. Clones containing gastrin sequences were selected by hybridization to a purified single-stranded 32P-labeled gastrin cDNA probe. This probe was constructed with gastrinoma mRNA as template. As primer for the cDNA synthesis, we used a synthetic oligonucleotide mixture, d(AG-A-A-AG-T-C-C-A-T-C-C-A), corresponding to the gastrin-specific amino acid sequence Trp-Met-Asp-Phe. In this way we determined the nucleotide sequence of the entire coding region (303 nucleotides), the entire 3' untranslated region (102 nucleotides), and 8 nucleotides of the 5' untranslated region. A striking homology between parts of the coding region suggests that evolution of the gastrin gene has involved a gene duplication.

Amino Acid Sequence↗

PubMed2001

Analysis of Pseudomonas putida alkane-degradation gene clusters and flanking insertion sequences: evolution and regulation of the alk genes.

The Pseudomonas putida GPo1 (commonly known as Pseudomonas oleovorans GPo1) alkBFGHJKL and alkST gene clusters, which encode proteins involved in the conversion of n-alkanes to fatty acids, are located end to end on the OCT plasmid, separated by 9.7 kb of DNA. This DNA segment encodes, amongst others, a methyl-accepting transducer protein (AlkN) that may be involved in chemotaxis to alkanes. In P. putida P1, the alkBFGHJKL and alkST gene clusters are flanked by almost identical copies of the insertion sequence ISPpu4, constituting a class 1 transposon. Other insertion sequences flank and interrupt the alk genes in both strains. Apart from the coding regions of the GPo1 and P1 alk genes (80-92% sequence identity), only the alkB and alkS promoter regions are conserved. Competition experiments suggest that highly conserved inverted repeats in the alkB and alkS promoter regions bind ALKS:

Alkanes↗

PubMed2002

Amplification-mutagenesis: evidence that "directed" adaptive mutation and general hypermutability result from growth with a selected gene amplification.

When a particular lac mutant of Escherichia coli starves in the presence of lactose, nongrowing cells appear to direct mutations preferentially to sites that allow growth (adaptive mutation). This observation suggested that growth limitation stimulates mutability. Evidence is provided here that this behavior is actually caused by a standard Darwinian process in which natural selection acts in three sequential steps. First, growth limitation favors growth of a subpopulation with an amplification of the mutant lac gene; next, it favors cells with a lac(+) revertant allele within the amplified array. Finally, it favors loss of mutant copies until a stable haploid lac(+) revertant arises and overgrows the colony. By increasing the lac copy number, selection enhances the likelihood of reversion within each developing clone. This sequence of events appears to direct mutations to useful sites. General mutagenesis is a side-effect of growth with an amplification (SOS induction). The F' plasmid, which carries lac, contributes by stimulating gene duplication and amplification. Selective stress has no direct effect on mutation rate or target specificity, but acts to favor a succession of cell types with progressively improved growth on lactose. The sequence of events--amplification, mutation, segregation--may help to explain both the origins of some cancers and the evolution of new genes under selection.

Electrophoresis, Agar Gel↗

PubMed1989

Evaluation of evolutionary divergence in the genus Naegleria by analysis of ribosomal DNA plasmid restriction patterns.

Ribosomal DNA (rDNA) plasmid restriction maps of 10 strains and rDNA hybridisation patterns of 61 additional strains have been used to assess inter- and intra-specific diversity and phylogenetic relationships in the genus Naegleria. The results obtained by this method largely confirm those of previous studies based on a variety of other criteria. They indicate that very little variation exists within the pathogenic species Naegleria fowleri despite its worldwide distribution and that it is closely related to the nonpathogenic Naegleria lovaniensis. Naegleria gruberi is most likely a polyphyletic grouping and care should be taken when using one strain as a reference point for this species. In addition, the two subspecies of the pathogenic Naegleria australiensis arose separately from within the range of variability encompassed by N. gruberi, as did the species Adelphamoeba galeacystis which should probably be assigned to the genus Naegleria. The species Naegleria jadini and Naegleria andersoni are not closely related to any other in the genus based on their rDNA patterns.

Animals↗

PubMed2005

In vitro selection and characterization of a stable subdomain of phosphoribosylanthranilate isomerase.

The (beta(alpha))8-barrel is the most common enzyme fold and it is capable of catalyzing an enormous diversity of reactions. It follows that this scaffold should be an ideal starting point for engineering novel enzymes by directed evolution. However, experiments to date have utilized in vivo screens or selections and the compatibility of (beta(alpha))8-barrels with in vitro selection methods remains largely untested. We have investigated plasmid display as a suitable in vitro format by engineering a variant of phosphoribosylanthranilate isomerase (PRAI) that carried the FLAG epitope in active-site-forming loop 6. Trial enrichments for binding of mAb M2 (a mAb to FLAG) demonstrated that FLAG-PRAI could be identified from a 10(6)-fold excess of a FLAG-negative competitor in three rounds of in vitro selection. These results suggest PRAI as a useful scaffold for epitope and peptide grafting experiments. Further, we constructed a FLAG-PRAI loop library of approximately 10(7) clones, in which the epitope residues most critical for binding mAb M2 were randomized. Four rounds of selection for antibody binding identified and enriched for a variant in which a single nucleotide insertion produced a truncated (beta(alpha))8-barrel consisting of (beta(alpha))1-5beta6. Biophysical characterization of this clone, trPRAI, demonstrated that it was selected because of a 21-fold increase in mAb M2 affinity compared with full-length FLAG-PRAI. Remarkably, this truncated barrel was found to be soluble, structured, thermostable and monomeric, implying that it represents a genuine subdomain of PRAI and providing further evidence that such subdomains have played an important role in the evolution of the (beta(alpha))8-barrel fold.

Aldose-Ketose Isomerases↗

PubMed1997

The evolution of beta-lactamases.

beta-lactamases, the enzymes often associated with resistance to beta-lactam antibiotics, are found in most bacterial species. Although these enzymes protected bacteria from naturally occurring beta-lactams long before the introduction of synthetic antimicrobial agents, the numbers and varieties of beta-lactamases have increased dramatically with the introduction of modern penicillins and cephalosporins. Over the past twenty years it has become apparent that families of beta-lactamases have been selected as the result of antimicrobial usage. Outbreaks of beta-lactam-resistant bacteria can be traced to the introduction of specific classes of beta-lactams or to the introduction of a specific agent. Many of the most serious epidemics can be related to transferable beta-lactamase genes that are harboured on multidrug-resistant plasmids. The separation of beta-lactamases into three major functional groups or four structural classes has been proposed. Stepwise selection of variants within several of these classes has been documented both in the clinical setting and in the laboratory, e.g. the extended-spectrum (TEM and SHV) beta-lactamases and the inhibitor-resistant (TEM) beta-lactamases. Close relationships among the recently described plasmid-mediated 'cephamycinases' and the common chromosomal cephalosporinases have been identified. Carbapenem-hydrolysing metallo-beta-lactamases with broad spectrum hydrolysing activity have become serious concerns as they begin to be described on plasmids. Factors contributing to selection of beta-lactam-resistant strains include decreased outer membrane permeability and increased beta-lactamase production.

Bacteria↗

PubMed1999

A homologue of the Agrobacterium tumefaciens VirB and Bordetella pertussis Ptl type IV secretion systems is essential for intracellular survival of Brucella suis.

Analysis of a TnblaM mutant of Brucella suis 1330, identified as being unable to multiply in Hela cells, allowed us to identify a 11 860 bp region of the B. suis genome encoding a type IV secretion system, homologous to the VirB system of Agrobacterium tumefaciens and the Ptl system of Bordetella pertussis. DNA sequence revealed 12 open reading frames (ORFs) encoding homologues of the 11 VirB proteins present in the pTi plasmid of Agrobacterium with a similar genetic organization, and a twelfth ORF encoding a putative lipoprotein, homologous to a protein involved in mating pair formation during bacterial conjugation and to adhesins used by Pseudomonas species to bind to plant roots. Phylogenetic trees based on the sequences of VirB4 and VirB9 protein homologues suggest that evolution of the systems from DNA transfer towards protein secretion did not stem from a single event but that the protein secretion systems have evolved independently. Four independent mutants in virB5, virB9 or virB10 were highly attenuated in an in vitro infection model with human macrophages. The virulence was restored by complementation with a plasmid containing the full virB region. The virB region appears to be essential for the intracellular survival and multiplication of B. suis.

Agrobacterium tumefaciens↗

PubMed1997

Avoidance of palindromic words in bacterial and archaeal genomes: a close connection with restriction enzymes.

Short palindromic sequences (4, 5 and 6 bp palindromes) are avoided at a statistically significant level in the genomes of several bacteria, including the completely sequenced Haemophilus influenzae and Synechocystis sp. genomes and in the complete genome of the archaeon Methanococcus jannaschii. In contrast, there is only moderate avoidance of palindromes in the small genome of the bacterium Mycoplasma genitalium and no detectable avoidance in the genomes of chloroplasts and mitochondria. The sites for type II restriction-modification enzymes detected in the given species tend to be among the most avoided palindromes in a particular genome, indicating a direct connection between the avoidance of short oligonucleotide words and restriction-modification systems with the respective specificity. Palindromes corresponding to sites for restriction enzymes from other species are also avoided, albeit less significantly, suggesting that in the course of evolution bacterial DNA has been exposed to a wide spectrum of restriction enzymes, probably as the result of lateral transfer mediated by mobile genetic elements, such as plasmids and prophages. Palindromic words appear to accumulate in DNA once it becomes isolated from restriction-modification systems, as demonstrated by the case of organellar genomes. By combining these observations with protein sequence analysis, we show that the most avoided 4-palindrome and the most avoided 6-palindrome in the archaeon M.jannaschii are likely to be recognition sites for two novel restriction-modification systems.

Archaea↗