Somatic cell mapping of the adenine nucleotide translocator gene family in cattle.
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Animals↗
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Nucleotide sequence and transcriptional mapping of the major capsid protein gene of pseudorabies virus.
The gene encoding the 142-kDa major capsid protein (MCP142) of pseudorabies virus (PrV) was isolated and sequenced. Nucleotide sequence analysis revealed that the MCP142 gene has a single open reading frame of 3993 nucleotides (nt) encoding 1330 amino acids. The 4400-nt major RNA from the MCP142 gene was detected in PrV-infected cells. The 5' end of the transcript was located 60 nt upstream of the initiation codon. The 3' end of the transcript was located 18 nt downstream of a putative poly(A) signal sequence TATAAA and 133 nt downstream of the termination codon. In comparing amino acid sequence homology between MCP142 of PrV and other available herpesviruses MCP was shown to have 58% homology with herpes simplex virus type 1 and varicella-zoster virus, 27% with Epstein-Barr virus, and 24% with human herpesvirus 6 and human cytomegalovirus. It has greater homology with those of the alpha-herpesviruses than with those of the beta-herpesviruses and the gamma-herpesviruses.
Amino Acid Sequence↗
Nucleotide sequence-directed mapping of the nucleosomes of SV40 chromatin.
In our previous work we have shown by comparison of experimental and computational data that the positions of the histone octamers bound to the DNA molecule appear to be completely sequence-dependent. This provides a convenient and quick method for locating the nucleosomes along the DNA molecule, as soon as the nucleotide sequence is known. Using this computational approach, the complete nucleosomal map of the SV40 minichromosome has been constructed. The map consists of 25 nucleosomes, with their coordinates (centers) being specified with high accuracy. The map is found to be in remarkable agreement with available experimental data.
Base Sequence↗
Characterization of the discontinuities in rice tungro bacilliform virus DNA.
The dsDNA of rice tungro bacilliform virus (RTBV) has two discontinuities, one on each strand, each in a specific position as found in other pararetroviruses. The 5' end of discontinuity 1 was mapped to nucleotide 1 of the published RTBV DNA sequence which suggests that tRNAiMet serves as a primer for negative strand DNA synthesis. This 5' terminus contains up to two ribonucleotides and the 3' terminus overlaps it by five to 25 nucleotides. The discontinuity 2 (D2) did not map to a purine-rich region as has been found in other similar viruses. Both the 5' and 3' termini of D2 were heterogeneous in position giving structures varying from a gap of 10 nucleotides to an overlap of 103 nucleotides.
Base Sequence↗
Pyrimidine (6-4) pyrimidone photoproduct mapping after sublethal UVC doses: nucleotide resolution using terminal transferase-dependent PCR.
UVC irradiation of genomic DNA induces two main types of potentially mutagenic base modifications: cyclobutane pyrimidine dimers (CPDs) and the less frequent (15-30% of CPD levels) pyrimidine (6-4) pyrimidone photoproducts (6-4PP). Ligation-mediated PCR (LMPCR), a genomic sequencing technique, allows CPD mapping at nucleotide resolution following irradiation with sublethal doses of UVB or UVC for most cell types. In contrast, a dose of 80 J/m(2) of UVC that is lethal for the majority of cell types is necessary to map 6-4PP by the LMPCR technique. This compromises the use of LMPCR to study the repair of 6-4PP. To date, no other techniques have been developed to study 6-4PP repair at nucleotide resolution. We have therefore adapted a recently developed technique for the mapping of 6-4PP: terminal transferase-dependent PCR (TDPCR). TDPCR is in many ways similar to LMPCR. This technique is more sensitive and allows the mapping of 6-4PP at UVC doses as low as 10 J/m(2) in genomic DNA and in living cells.
Binding Sites↗
NAD(+)-specific glutamate dehydrogenase of Neurospora crassa: cloning, complete nucleotide sequence, and gene mapping.
The NAD(+)-specific glutamate dehydrogenase (NAD-GDH) of the filamentous fungus Neurospora crassa is a tetrameric enzyme, regulated by catabolite repression. The amino acid sequence of this enzyme had been published several years ago. With the object of investigating the molecular mechanism of catabolite repression, the nucleotide sequence of genomic clones containing the coding region, along with 5'- and 3'-flanking noncoding segments of the NAD-GDH transcription unit, was obtained. The gdh structural gene was shown to code for a polypeptide of 1047 residues, with a calculated molecular mass of 118,280 daltons. The coding sequence is interrupted by two short introns located close to the N- and C-terminal domains of the polypeptide. Consensus intron boundaries and internal splice sequences resemble closely those of other N. crassa genes. A comparison of the amino acid sequence deduced from the nucleotide sequence with the previously published sequence showed several discrepancies between the two. Nucleotide sequence corresponding to a gap in the amino acid sequence was located in the genomic clone. Genetic mapping by restriction fragment length polymorphism analysis localized the gdh gene close to the loci trp-1 and con-7 on the right arm of linkage group III.
Amino Acid Sequence↗
Changes in the topography of early region transcription during polyoma virus lytic infection.
We have studied the pattern of transcription of the early region of polyoma virus DNA after the onset of the late phase of lytic infection of mouse cells. Following initiation of viral DNA synthesis, the early/late switch is accompanied not only by efficient production of late mRNAs but also by the appearance of previously unidentified early-strand RNAs which have certain structural features in common with the classical early mRNAs. Stable poly(A)+RNAs have been identified by blot analysis and S1 nuclease mapping that are not detected early during infection or in polyoma virus-transformed cells. One group consists of transcripts whose 5' ends map 150-200 nucleotides upstream from the major early 5' ends (at positions 148 and 153 on the polyoma virus genome) but whose splicing pattern and poly(A) addition sites are indistinguishable from those of mRNAs produced early in infection. The 5' exons of these early region transcripts contain an open translational reading frame that extends from nucleotide positions 5,255 to 124 and is capable of encoding a basic protein of 53 amino acids. Transcription of these RNAs does not appear to be negatively regulated by large tumor antigen. A transcript of 1,800 nucleotides appears to map predominantly between 93 and 26 map units and does not contain sequences present in the early mRNA 5' exons. These data suggest that, after the onset of polyoma DNA replication, the activation of new early-strand promoters leads to the expression of previously untranscribed viral DNA sequences.
Antigens, Viral↗
Single nucleotide polymorphisms for integrative mapping in the Turkey (Meleagris gallopavo).
When multiple genetic maps exist for a species, integration of these maps requires a set of common markers be genotyped across the individual mapping populations. In the turkey, three genetic maps based on separate mapping populations are available. In this study, SNP-based markers were developed for integrating the cDNA/RFLP-based map (1) with microsatellite markers of the second-generation turkey genome map (2). Forty-eight primer sets were designed and tested and 33 (69%) correctly amplified turkey genomic DNA by PCR. Putative SNPs were detected in 20 (61%) of the amplified gene fragments, and 10 SNP markers were subsequently genotyped by PCR/RFLP for segregation analysis. Eight SNP markers were incorporated into the turkey genetic map.
Animals↗
DNA fingerprints of poultry.
Human minisatellite probes cross-hybridize to DNA of several species of poultry (chicken, duck, turkey and goose), and detect high levels of polymorphism. The resulting DNA fingerprints are individual specific, and allow the discrimination even between closely related birds. The pattern of poultry DNA fingerprints is different from that of humans and other animals, having a higher average proportion of large DNA fragments. Pedigree analysis revealed a low number of allelic pairs of variable DNA fragments, indicating that most of the alleles are unresolved in the DNA fingerprint or too small to be detected. The total number of detectable loci in broilers, using probe 33.6, was estimated as 62, of which 13 loci are on average scoreable and available for use. Poultry DNA fingerprints can be used for individual identification, linkage studies and as an aid in breeding programmes.
Alleles↗
Genes of the Escherichia coli pur regulon are negatively controlled by a repressor-operator interaction.
Fusions of lacZ were constructed to genes in each of the loci involved in de novo synthesis of IMP. The expression of each pur-lacZ fusion was determined in isogenic purR and purR+ strains. These measurements indicated 5- to 17-fold coregulation of genes purF, purHD, purC, purMN, purL, and purEK and thus confirm the existence of a pur regulon. Gene purB, which encodes an enzyme involved in synthesis of IMP and in the AMP branch of the pathway, was not regulated by purR. Each locus of the pur regulon contains a 16-base-pair conserved operator sequence that overlaps with the promoter. The purR product, purine repressor, was shown to bind specifically to each operator. Thus, binding of repressor to each operator of pur regulon genes negatively coregulates expression.
Base Sequence↗
Nucleotide sequence and chromosomal mapping of the human c-yes-2 gene.
We molecularly characterized the second gene, c-yes-2, of two copies of yes-related genes which we previously found to contain in the human genome. First, nucleotide sequence analysis revealed that the c-yes-2 gene is a pseudogene of the c-yes-1 gene. Second, by using two independent methods, hybridization of both DNAs from sorted chromosomes and metaphase spreads with c-yes-2 DNA, we assigned the c-yes-2 gene to chromosome 22q11.2. This chromosomal localization is consistent with that given in our previous report. The failure of proper mapping in our experiment might have been caused by instability of hybrid cell clones.
Base Sequence↗
Nucleotide sequence and revised map location of the arn gene from bacteriophage T4.
Non-glucosylated (Glu-) T-even phage DNAs are restricted by Escherichia coli RgIA and RgIB endonucleases with different specificities. RgIB endonuclease activity is strongly inhibited by anti-restriction endonuclease (Arn) encoded by the bacteriophage T4 genome. The nucleotide sequence of the arn gene encoding Arn was determined. The product of the cloned arn gene was overexpressed by the T7 RNA polymerase/promoter system, and its molecular size is consistent with that predicted from the open reading frame of the arn gene. The arn gene is located between the asiA gene and motA gene in the region of 161,300-161,578 nucleotides.
Amino Acid Sequence↗
Unique temperature-sensitive defect in vaccinia virus morphogenesis maps to a single nucleotide substitution in the A30L gene.
Marker rescue experiments demonstrated that the genetic lesion of a previously isolated vaccinia virus temperature-sensitive mutant which forms multilayered envelope structures with lucent interiors and foci of viroplasm with dense centers mapped to the A30L open reading frame. A single base change, resulting in a nonconservative Ser-to-Phe substitution at residue 17, was associated with degradation of the A30L protein at elevated temperatures.
Base Sequence↗
Linkage mapping of a single nucleotide polymorphism (SNP) in the porcine QDPR gene to chromosome 8.
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Animals↗
Nucleotide sequence and functional map of pE194, a plasmid that specifies inducible resistance to macrolide, lincosamide, and streptogramin type B antibodies.
pE194 is a small plasmid (isolated originally in Staphylococcus aureus) which confers erythromycin-inducible resistance to macrolide, lincosamide, and streptogramin type B (MLS) antibiotics. The nucleotide sequence of pE194 contains 3,728 base pairs (bp), corresponding to a molecular mass of 2.4 million daltons. By means of site-specific cleavage with restriction endonucleases and cloning resultant fragments, determinants of the two major biological functions of p E194, i.e., inducible MLS resistance and replication, could be localized and assigned to specific sequences in the plasmid. Restriction endonuclease TaqI cut pE194 at three sites. TaqI fragment A (1,443 bp) contained the determinant for inducible MLS resistance, whereas TaqI fragment B (1,354 bp) contained a determinant necessary for plasmid replication. Regulatory mutations resulting in constitutive expression of MLS resistance mapped in TaqI fragment A, whereas a mutation associated with elevated plasmid copy number was mapped in TaqI fragment B. Also mapping in TaqI fragment B was a plasmid replication determinant comprising two sets of inverted complementary repeat sequences, one of which spanned 124 bp and was adjacent to a second smaller set which was rich in guanine and cytosine residues. pE194 contained six open reading frames which were theoretically capable of coding for proteins with maximum molecular masses as follows (in daltons): A, 48,300; B, 29,200; C, 14,000; D, 13,900; E, 12,600; and F, 2,700. Insertion of plasmid pBR322 into the single PstI site located in frame A of pE194 resulted in a composite plasmid which could replicate in both Bacillus subtilis and Escherichia coli, suggesting that an intact polypeptide A is dispensable for both replication of pE194 and for MLS resistance. Frame B specified inducible MLS resistance, whereas frame F specified the putative peptide associated with the proposed B determinant translational attenuator. The extent to which frames C, D, and E, all contained in TaqI fragment B, were translated into polypeptide products is not known; however, a base change in frame E was found in a comparison between the high-copy-number mutant, cop-6, and the wild-type strains.
Amino Acid Sequence↗
S1 mapping of purified nascent transcripts of simian virus 40.
We purified nascent simian virus 40 late transcripts by incubating viral transcriptional complexes, isolated from infected BSC-1 cells, in a reaction mixture that contained mercurated CTP; RNA molecules that had incorporated mercurated residues in vitro were isolated by sulfhydrylcellulose affinity chromatography. The nascent RNA was hybridized to an end-labeled HindIII C probe fragment (0.646 to 0.86 map unit), and the hybrids were analyzed by S1 mapping. Most of the products of digestion corresponded to unspliced transcripts with 5' ends mapping at nucleotides 325, 260, and 195, which are positions of the 5' ends of mature, cytoplasmic late mRNA species. In addition, two minor products diagnostic of splicing at the acceptor junctions mapping at nucleotides 556 and 443 were detected. Because the abundance of these products was not diminished by repurifying the nascent RNA through a second round of sulfhydrylcellulose chromatography, these products did not originate from contaminating non-nascent RNA. Moreover, the generation of these products was not affected when a higher salt concentration and lower temperature were used for S1 digestion, conditions that should decrease artifactual cleavage by S1 in A + U-rich regions of colinear hybrids. Therefore, it is likely that some simian virus 40 RNA chains are spliced before release from the template.
Base Sequence↗
Dynamics of the microtubule oscillator: role of nucleotides and tubulin-MAP interactions.
Microtubules can be induced to perform synchronous and periodic cycles of assembly and disassembly at constant temperature. The process depends on GTP hydrolysis. Time-resolved X-ray scattering using synchrotron radiation shows a cyclic interconversion of tubulin subunits, microtubules and oligomers (= short protofilament fragments). Oscillations are correlated with conditions that stabilize polymers and destabilize oligomers, and others of opposite effect. Microtubule stabilizers include GTP, Mg2+ or microtubule-associated proteins (MAPs), destabilizers include GDP or elevated ionic strength. K+ at intracellular concentrations noticeably increases the stability of tubulin-MAP oligomers, in contrast to Na+. ATP and the non-hydrolyzable analogue AMP-PNP enhance oscillations by mechanisms that are not directly linked to the role of nucleotide hydrolysis in assembly. We propose a mechanism of oscillations that include oligomers as microtubule disassembly products which transiently lock the protein in an unpolymerizable state; this may point to a role of oligomers in controlling microtubule assembly cycles in cells.
Adenosine Triphosphate↗
Cloning, nucleotide sequencing, and genetic mapping of the gene for small, acid-soluble spore protein gamma of Bacillus subtilis.
The Bacillus subtilis gene (sspE) which codes for small acid-soluble spore protein gamma (SASP-gamma) was cloned, and its chromosomal location (65 degrees, linked to glpD) and nucleotide sequence were determined. The amino acid sequence of SASP-gamma is similar to that of SASP-B of Bacillus megaterium, but these sequences are not as highly conserved across species as are those of other SASPs. The SASP-gamma gene is transcribed only in sporulation in parallel with other SASP genes and gives a single mRNA that is approximately 340 nucleotides long. The results of hybridization of an sspE gene probe to Southern blots of B. subtilis DNA suggested that there is only a single gene coding for the SASP-gamma type of protein in B. subtilis. This was confirmed by introducing a deletion mutation into the cloned sspE gene and transferring the deletion into the B. subtilis chromosome, with concomitant loss of the wild-type gene. This sspE deletion strain sporulated well, but lacked the SASP-gamma type of protein.
Amino Acid Sequence↗