Non-homologous Regions Research Articles

Nucleotide sequence of the cell wall proteinase gene of Streptococcus cremoris Wg2.

A 6.5-kilobase HindIII fragment that specifies the proteolytic activity of Streptococcus cremoris Wg2 was sequenced entirely. The nucleotide sequence revealed two open reading frames (ORFs), a small ORF1 with 295 codons and a large ORF2 containing 1,772 codons. For both ORFs, there was no stop codon on the HindIII fragment. A partially overlapping PstI fragment was used to locate the translation stop of the large ORF2. The entire ORF2 contained 1,902 coding triplets, followed by an apparently rho-independent terminator sequence. The inferred amino acid sequence would result in a protein of 200 kilodaltons. Both ORFs have their putative transcription and translation signals in a 345-base-pair ClaI fragment. ORF2 is preceded by a promoter region containing a 15-base-pair complementary direct repeat. Both the truncated 33- and the 200-kilodalton proteins have a signal peptide-like N-terminal amino acid sequence. The protein specified by ORF2 contained regions of extensive homology with serine proteases of the subtilisin family. Specifically, amino acid sequences involved in the formation of the active site (viz., Asp-32, His-64, and Ser-221 of the subtilisins) are well conserved in the S. cremoris Wg2 proteinase. The homologous sequences are separated by nonhomologous regions which contain several inserts, most notably a sequence of approximately 200 amino acids between the His and Ser residues of the active site.

Homology of lysosomal enzymes and related proteins: Prediction of posttranslational modification sites including phosphorylation of mannose and potential epitopic and substrate binding sites in the α‐ and β‐subunits of hexosaminidases, α‐glucosidase, and rabbit and human isomaltase

Recently developed computer programs, including secondary structure and epitopic site predictions, have been used to align lysosomal proteins for maximum homology, based on conservative interchanges, and the aligned sequences have been searched for potential sites for posttranslational modification, glycosylation, and binding and catalysis of substrate. The homology and prediction of the posttranslational modification of the alpha- and beta-subunits of hexosaminidase is in good agreement with previous observations, and an explanation of the differing substrate specificities of the two subunits is advanced. We show that the striking homology between alpha-glucosidase and isomaltase is reflected in the apparent conservation of the active site in both enzymes. Nonhomologous regions have been examined in detail in a search for binding sites for glycogen and maltose, and two such sites have been tentatively identified. A highly redundant consensus sequence for the phosphorylation of mannose in lysosomal proteins, YXX(Y, W, or F), is suggested.

Complementary DNA cloning of cytochrome P-450s related to P-450(M-1) from the complementary DNA library of female rat livers. Predicted primary structures for P-450f, PB-1, and PB-1-related protein with a bizarre replacement block and their mode of transcriptional expression.

Three kinds of cDNA clones were isolated from a cDNA library synthesized from female rat liver mRNA by cross-hybridization with the P-450(M-1) cDNA as a probe and sequenced. One clone appears to be the previously isolated P-450f cDNA clone with an additional 5'-untranslated and coding sequence which are lacking in the previously reported clone (Gonzalez, F. J., Kimura, S., Song, B.-J., Pastewka, J., Gelboin, H. V., and Hardwick, J. P. (1986) J. Biol. Chem. 261, 10667-10672), though several nucleotide differences were seen. Another one is for P-450PB-1 mRNA previously isolated, and the last has an almost identical nucleotide sequence to P-450PB-1 (the same report cited above) except for one region of 159 base pairs where the sequence homology between the two is abruptly broken down. This nonhomologous region appears to correspond exactly to the entire eighth exon, estimated by comparison with the gene structure of the related P-450 (P-450(M-1)). This replacement in P-450PB-1 (ps) causes a frameshift in the open reading frame, resulting in the generation of a truncated form of P-450 with a strange replacement block and lacking the heme-binding region. This observation suggests that the mRNA whose cDNA was cloned here was produced from a recombinant gene generated by gene conversion or from alternative splicing of a cryptic exon. Sex- and age-dependent expression of the mRNAs investigated by dot blot analysis revealed that normal- and pseudo-type PB-1 mRNA were expressed in both male and female rat livers, though their age-dependent expression was different in male and female animals. In addition, both the mRNAs were specifically expressed in the female brain of 8 weeks, whereas practically no expression was observed in kidney and lung of both sexes.

O-antigen variation in Salmonella spp.: rfb gene clusters of three strains

The O antigens of Salmonella serogroups A, B, and D differ structurally in their side-chain sugar residue. These genes encoding O-antigen biosynthesis are clustered in the rfb operon. We report here the molecular cloning and analysis of the rfb operons of Salmonella paratyphi A (serogroup A) and S. typhi (serogroup D). The regions of DNA nonhomology between the rfb operons of these serogroup A, B, and D representatives are identified, and the evolutionary derivation of serogroup A from a serogroup D progenitor is discussed.

The chicken c-fos gene: cloning and nucleotide sequence analysis.

Using a DNA probe from an avian transforming virus, NK24, that contains the fos gene and that was newly isolated and characterized (M. Nishizawa, N. Goto, and S. Kawai, J. Virol. 61:3733-3740, 1987), we cloned and sequenced the chicken c-fos gene. Results showed that this gene, like the mouse or human c-fos gene, contains four exons encoding a predicted gene product of 367 amino acids which is about 79% homologous to p55c-fos (mouse), excluding several small insertions or gaps. The predicted gene product, however, contains two nonhomologous regions, as suggested by sequence analysis of the NK24 genome. About half the identical amino acids of these gene products are encoded by different codons, and the guanine-plus-cytosine content in the third letter of each codon used in the entire chicken c-fos coding region is high (93%). The 5'-flanking region of the TATA box contains a sequence that is homologous to a putative transcriptional regulatory sequence in the mouse or human c-fos gene. The 3'-noncoding region of chicken c-fos is strikingly homologous to that of mouse or human c-fos and covers a 67-base-pair adenine-plus-thymine-rich stretch which was previously reported to be essential for an inhibitory effect on the expression of the mouse c-fos gene.

Mismatch repair during pneumococcal transformation of small deletions produced by site-directed mutagenesis.

The genetic behaviour of short non-homologous regions has been studied during transformation of Streptococcus pneumoniae. Amethopterin-resistant mutants belonging to the amiA locus were used for these investigations. Five mutants deleted for 1-5 bp were obtained by oligonucleotide-directed mutagenesis. Their efficiency of transformation was measured using recipient strains either able to excise and repair mismatched bases (Hex+) or Hex- derivatives. Deletions or insertions of 1 and 2 bp are fully recognized by the Hex system, and are efficiently repaired whereas 3-bp deletions or insertions are only partially excised and repaired. The efficiency of repair is inversely related to the size of the non-homology. Markers with 5-bp deletions or insertions are poorly repaired and thus transform at very high frequency: similar results are obtained in reciprocal crosses. It is proposed that 1- or 2-bp deletions or insertions are included in the heteroduplex structure as transition mutations. The Hex system would detect only small deviations from the normal DNA structure.

High degree of homology of the deduced protein structures of the tetracycline-resistance determinants between Bacillus subtilis plasmid pNS1981 and Staphylococcus aureus plasmid pTP5

The amino acid sequences of the tetracycline-resistance (Tcr) determinants of Bacillus subtilis plasmid pNS1981 and Staphylococcus aureus plasmid pTP5 have been deduced from their nucleotide sequences and compared. The deduced Tcr proteins (TETs) of pNS1981 (458 amino acids) and pTP5 (459 amino acids) show a considerable homology (60% identical). If homologous amino acid replacement is taken into account, the homology becomes 80%. Both TET proteins are highly hydrophobic, as expected for a membrane-binding protein, and their polarities are calculated at 32–33%. The putative secondary structures of both TET proteins have been also shown to be significantly homologous, being abundant in β-sheets. The predicted positions of β-sheets show a nice coincidence between both TET proteins. α-Helix has a tendency to be formed at nonhomologous regions of the primary structures between both TET proteins. However, the predicted positions of α-helices coincide in a frequency greater than 50%. α-Helix and random coil moderately occur at the hydrophilic regions in both TET proteins.

A sex chromosome rearrangement in a human XX male caused by Alu—Alu recombination

Human XX maleness is often due to the presence of Y-specific DNA, resulting from abnormal interchange of terminal parts of the short arms of the X and Y chromosomes. In an XX male, a rearrangement is observed at locus DXYS5, the most proximal Yp locus detected in this patient. Cloning and analysis of the rearranged DNA fragment revealed pseudoautosomal sequences located beyond the breakpoint. We propose that this XX male arose by abnormal crossing over between DXYS5 on the Y chromosome and a pseudoautosomal locus on the X chromosome during paternal meiosis. Sequence analysis of the junction shows that homologous recombination occurred between two Alu sequences from these otherwise nonhomologous regions. The site of recombination is localized to the putative transcription promoter region of the Alu sequences.

OriT sequence of the antibiotic resistance plasmid R100.

We present the nucleotide sequence of the oriT region from plasmid R100. Comparison to other IncF plasmids revealed homology around the proposed nick sites as well as conservation of inverted repeated sequences in the nonhomologous region. Three areas showed strong homology (eight of nine nucleotides) to the consensus sequence for binding of integration host factor, suggesting a role for this DNA-binding protein in nicking at oriT.

Conservation and variation in orf virus genomes

The genomes of several orf virus strains were analyzed with the restriction endonucleases EcoRI, HindIII, BamHI, and KpnI, and cleavage site maps were deduced. In general, the right half of the genome showed conservation of restriction sites compared with the left half. Variations in size of up to 0.5 kbp were found within an inverted terminal repetition, and a 1-kbp deletion was detected in some strains in a subterminal fragment at the left end. A region of approximately 20 kbp, some 12 kbp in from the left end, showed the greatest cleavage site variability although there was no evidence of large deletions in this region. A 1.55-kbp cloned DNA fragment from the internal variable region of NZ2 failed to hybridize to the DNA from three other strains. A fragment in the variable region of strain NZ7 was cloned and compared by hybridization and restriction endonuclease analysis with cloned NZ2 fragments from the same region. The region of nonhomology extended for at least 2.75 kbp. It is suggested that this internal variable region may provide sites for the insertion of foreign genes.

Gonadotropin and thyrotropin alpha- and beta-subunit gene expression in normal and neoplastic tissues characterized using specific messenger ribonucleic acid hybridization probes.

The glycoprotein hormones consist of a common alpha-subunit and distinct, but structurally related, beta-subunits which confer biological specificity. To study glycoprotein hormone gene expression, we prepared specific oligonucleotides complementary to nonhomologous regions of the alpha-subunit and each of the beta-subunit mRNAs encoding human LH, CG, TSH, and FSH. beta-Subunit mRNAs were expressed at relatively low levels in normal pituitary tissue, but were found in greater amounts in pituitary gonadotroph and thyrotroph adenomas. The lengths of the glycoprotein hormone alpha- and beta-subunit mRNAs in normal and neoplastic pituitary tissue were indistinguishable. Expression of different members of the closely related LH beta/CG beta gene family were examined in normal and neoplastic pituitary and placenta using short oligonucleotides complementary to nonhomologous regions of the genes. Although CG beta mRNA was found previously in a pituitary adenoma, none was detected in normal pituitary tissue. In placenta, there was abundant expression of the CG beta gene, but no expression of the LH beta gene, consistent with the acquisition of tissue-specific regulatory sequences in the recently evolved upstream promoter recognition site of the CG beta gene. Primer extension analysis of CG beta mRNA indicated that the same CG beta gene promoter site was used in both normal placenta and JEG-3 choriocarcinoma cells. Of the two CG beta genes that have been reported to be functional, CG beta gene 5 was preferentially expressed in both normal placenta and the neoplastic JEG-3 cell line. CG beta gene 3 expression accounted for only about 5% of the total CG beta mRNA. The previously uncharacterized human TSH beta and FSH beta mRNAs were studied in normal and neoplastic pituitary tissue. At least two species of FSH beta mRNA were found on Northern blots. Oligonucleotide-primed extension of pituitary mRNA demonstrated that FSH beta mRNA heterogeneity resulted from transcription from distinct promoter sites, encoding 5'-untranslated tracts of 48 and 83 bases. These studies demonstrate that specific oligonucleotide probes distinguish expression of the structurally related glycoprotein hormone beta-subunit mRNAs, allowing analyses of tissue-specific gene expression under different physiological conditions as well as in normal and neoplastic tissues.

A unique DNA structure of the junction of homologous and nonhomologous regions between tetracycline-resistance plasmid pNS1981 and kanamycin-resistance plasmid pUB110.

A unique DNA structure of the junction of homologous and nonhomologous regions between tetracycline-resistance plasmid pNS1981 and kanamycin-resistance plasmid pUB110.

Two DNA recognition domains of the specificity polypeptides of a family of type I restriction enzymes.

The hsd genes of Salmonella typhimurium and Salmonella potsdam encode related type I restriction and modification systems designated SB and SP, respectively; the polypeptide encoded by the hsdS gene dictates the DNA sequence recognized. The hsdS genes of the SB and SP systems have a conserved sequence of around 100 base pairs flanked by two nonhomologous (variable) regions of around 500 base pairs. Recombination between the hsdS genes of SB and SP generated a system (SQ) with a different recognition specificity. We have localized the position of the crossover in the central conserved region by analysis of nucleotide sequences. Concomitant with the generation of a new combination of flanking variable regions is the recombination of minor differences in the central conserved region. A polypeptide domain encoded on the 5' side of the crossover dictates recognition of the trinucleotide component of the target sequence, and a second domain, encoded on the 3' side of the crossover, similarly governs recognition of the tetra- or penta-nucleotide component. Our analysis implicates at least parts of the variable regions in the determination of the specificity of interaction between protein and DNA. Furthermore, the trinucleotide components of the recognition sequences of S. typhimurium and Escherichia coli K-12 are identical, and the 5' segments of their hsdS genes are strikingly homologous rather than variable.

Mitochondrial DNA restriction site variation in the Townsend's vole, Microtus townsendii

The mitochondrial DNA (mtDNA) from the Townsend's vole, Microtus townsendii, was compared with mouse mtDNA by positioning vole restriction enzyme fragments on the known laboratory mouse mtDNA molecule by homology hybridization. The vole mitochondrial genome is conserved in general sequence organization and size but does contain a nonhomologous region of more than 600 base pairs not found in the mouse sequence. Thirty-five voles were collected from seven different localities throughout the range of the species, including insular populations on Bowen and Vancouver islands. The variation of vole mtDNA sequences within this species was assayed with six hexameric and four tetrameric type II restriction endonucleases. These individuals can be divided into seven distinct maternal lines. The level of nucleotide substitution between populations is shown to be as high as 0.896 ± 0.350%. The voles from Bowen Island showed no detectable variation within their population, or divergence from a mainland population on the adjacent coast. This fact suggests a recent colonization of Bowen Island. The samples from Vancouver Island fall into two major maternal lines, which show 0.453 ± 0.240% divergence. These insular maternal lines are 0.677 ± 0.257% divergent from the most closely related mainland population. These results suggest that the voles on Vancouver Island represent long-established maternal lines, and are not derived by a recent colonization from a mainland source population. Based on conservative estimates for rates of nucleotide substitution, Vancouver Island has been inhabited by the Townsend's vole since at least the Olympic interglacial.

Isolation and characterization of the complete complementary and genomic DNA sequences of human serum amyloid P component.

Complementary and genomic DNA clones corresponding to the human serum amyloid P component (SAP) mRNA have been isolated and analyzed. The nucleotide sequences of the cDNA and the corresponding regions of the genomic SAP DNA reported here were identical, and revealed that after coding for a signal peptide of 19 amino acids and the first two amino acids of the mature SAP protein, there is one small intron of 115-base pairs (bp), followed by a nucleotide sequence coding for the remaining 202 amino acid residues. The SAP gene has an ATATAAA sequence 29-bp upstream from the cap site, but there is no CAAT box-like sequence. A possible polyadenylation signal sequence, ATTAAA, was found to be located 28-bp upstream from the polyadenylation site. A comparison of the genomic SAP DNA sequence with that of human C-reactive protein (CRP) revealed a striking overall homology which was not uniform: several highly conserved regions were bounded by non-homologous regions. This comparison provides further support for the hypothesis that SAP and CRP are products of a gene duplication event.

Sequence relations among the IncY plasmid p15B, P1, and P7 prophages

Electron microscopic analysis of heteroduplex molecules between the 94-kb plasmid p15B and the 92-kb phage P1 genome revealed nine regions of nonhomology, eight substitutions, and two neighboring insertions. Overall, the homologous segments correspond to 83% of the P1 genome and 81% of p 15B. Heteroduplex molecules between p 15B and the 99-kb phage P7 genome showed nonhomology in eight of the same nine regions; in addition, two new nonhomologous segments are present and P7 carries a 5-kb insertion representing Tn 902. The DNA homology between those two genomes amounts to 79% of P7 DNA and 83% of p15B. Plasmid p15B contains two stem-loop structures. One of them has no equivalent structure on P1 and P7 DNA. The other substitutes the invertible C segments of P1 and P7 and their flanking sequences including cin, the gene for the site-specific recombinase mediating inversion.

Recombination between IS5 elements: requirement for homology and recombination functions.

Intermolecular recombination between two IS5 elements was measured, using bacteriophage lambda recombination vectors, and was compared to recombination between two copies of an SV40 segment cloned into the same vectors. Experiments were conducted in the presence and in the absence of RecA and Red functions, and with the recombining inserts in the same or in reversed orientation. Under all conditions, IS5 elements recombined in a manner similar to the SV40 inserts, indicating that IS-encoded functions did not confer measurable additional intermolecular recombination ability to IS5 in E. coli K-12. Bacteriophages containing reversed IS5 inserts, for which the 16 base pair (bp) termini are identical in 15 positions and which display 12 bp of uninterrupted homology, recombined at approximately the same low frequency under Rec+ and Rec- conditions, indicating that these short homologies were not good substrates for the Rec system. Bacteriophages having reversed inserts recombined better under Red+ than under Red- conditions, but the crossovers were located in nonhomologous regions flanking the element termini. This suggests that 12-bp homologies are not good substrates for the Red system.

Distribution, inheritance and linkage relationships of ribosomal DNA spacer length variants in pea

DNA restriction endonuclease fragment analysis is used to examine the genetic organization, inheritance and linkage associations of the ribosomal DNA in pea. The substantial variation observed in the length of the intergenic spacer region is shown to segregate in Mendelian fashion involving two independent genetic loci, designated Rrn1 and Rrn2. Linkage between Rrn1 and two marker loci on chromosome 4 establishes the approximate location of this tandem array. Rrn2 shows linkage with a set of isozyme loci which assort independently of other markers on all seven chromosomes. Combining these observations with previous cytological data, we suggest that Rrn2 and the isozyme loci linked to it constitute a new linkage group on chromosome 7. The general absence of spacer length classes common to both rRNA loci in any of the lines we examined indicates that little or no genetic exchange occurs between the nonhomologous nucleolar organizer regions.

Genetic and molecular characterization of argB+ transformants of Aspergillus nidulans.

Thirty-three argB- to argB+ transformants of Aspergillus nidulans have been subjected to genetic and molecular analysis. Two showed high levels of mitotic instability although it is suggested that this is a consequence of heterokaryosis rather than instability of the transformation event. Most transformants resulted from the integration of the transforming DNA in tandem with the chromosomal argB locus. The maximum number of inserted sequences was two, to generate three copies of the argB locus. The other main transformant type showed replacement of the argB- mutation by the wild-type allele present on the transforming plasmid. Transformants were also recovered in which the transforming DNA had integrated into non-homologous chromosomal regions. Selfed or hybrid cleistothetica from all transformants, except the gene replacement types gave arginine requiring recombinants. Most transformants showed low levels of meiotic instability. Others displayed varying levels which in some cases differed between selfed and hybrid cleistotheticia. There was some correlation between meiotic instability and the nature of the transformation event. Diploid parasexual and aneuploid analysis located the integrated DNA in each transformant to chromosome III. Two transformants were isolated as heterozygous diploids. A third diploid was isolated as a stable mitotic segregant from one of the mitotically unstable transformants.

Characterization of apolipoprotein B-containing lipoproteins

Publisher Summary This chapter focuses on the means of preventing artifactual modification of apolipoprotein B (apoB)-containing lipoproteins during preparation and on selected aspects of their characterization. Characterization of intact lipoproteins involves the application of a number of physical and chemical methods. The chapter discusses the determination of particle size distribution. Electron microscopy of negatively stained preparations, assisted by computerized digitization, has proved a facile method for generating histograms of apparent particle diameter. In this technique, error caused by flattening of particles is difficult to correct precisely, but for all but the largest triglyceride-rich particles, it is probably of a low order. Study of electrophoretic heterogeneity of lipoproteins within a given ultracentrifugal density fraction has led to the discovery of important classes of lipoproteins. Methods employing immunosorption may become the most effective means of isolating subfractions of apoB-containing lipoproteins. Monoclonal antibodies to the nonhomologous region of apoB-100 allow complete separation, by column immunosorption, of chylomicrons and their remnants from very-low-density lipoproteins (VLDL).