共查询到20条相似文献,搜索用时 15 毫秒
1.
Summary We have determined the DNA sequence of a BALB/cTla region class I gene from the major histocompatibility complex (MHC) that had been identified previously as encoding a murine antigen by DNA-mediated gene transfer. Analysis of the DNA sequence shows, however, that this gene, the T1c gene from theTla
c genotype, could not encode a TL antigen or any other functional class I molecule due to the presence of numerous stop codons and frameshift mutations in the coding regions. This result suggests that the earlier transformation data may have been incorrect or perhaps that the clone containing the T1c gene contains sequences that induced expression of a serologically reactiveTla gene in the genome of the recipient L cell. The T1c gene is structurally related to the previously sequenced T13c gene that encodes a serologically defined TL antigen. The 3 half of the T1c gene including exons 4, 5, 6, and the 3 untranslated region has about 85% nucleotide similarity (including introns) with the corresponding parts of the T13c gene; however, the 5 half of the T1c gene has little homology with the T13c gene. There is a sharp line of demarcation between the homologous and nonhomologous regions, and this border occurs precisely at a B2 Alu repeat sequence present in the T13c gene. This suggests that a recombination event took place here and that an Alu repeat sequence that is known to have characteristics of transposable elements played some role in a recombination or gene conversion event. 相似文献
2.
The Chinese hamster dihydrofolate reductase replication origin beta is active at multiple ectopic chromosomal locations and requires specific DNA sequence elements for activity
下载免费PDF全文
![点击此处可从《Molecular and cellular biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
To identify cis-acting genetic elements essential for mammalian chromosomal DNA replication, a 5.8-kb fragment from the Chinese hamster dihydrofolate reductase (DHFR) locus containing the origin beta (ori-beta) initiation region was stably transfected into random ectopic chromosomal locations in a hamster cell line lacking the endogenous DHFR locus. Initiation at ectopic ori-beta in uncloned pools of transfected cells was measured using a competitive PCR-based nascent strand abundance assay and shown to mimic that at the endogenous ori-beta region in Chinese hamster ovary K1 cells. Initiation activity of three ectopic ori-beta deletion mutants was reduced, while the activity of another deletion mutant was enhanced. The results suggest that a 5.8-kb fragment of the DHFR ori-beta region is sufficient to direct initiation and that specific DNA sequences in the ori-beta region are required for efficient initiation activity. 相似文献
3.
A fully active variant of dihydrofolate reductase with a circularly permuted sequence. 总被引:13,自引:0,他引:13
The amino acid sequence of mouse dihydrofolate reductase was permuted circularly at the level of the gene. By transposing the 3'-terminal half of the coding sequence to its 5' terminus, the naturally adjacent amino and carboxyl termini of the native protein were fused, and one of the flexible peptide loops at the protein surface was cleaved. The steady-state kinetic constants, the dissociation constants of folate analogues, and the degree of activation by both mercurials and salt as well as the resistance toward digestion by trypsin were almost indistinguishable from those of a recombinant wild-type protein. Judged by these criteria, the circularly permuted variant has the same active site and overall structure as the wild-type enzyme. The only significant difference was the lower stability toward guanidinium chloride and the lower solubility of the circularly permuted variant. This behavior may be due to moving a mononucleotide binding fold from the interior of the sequence to the carboxyl terminus. Thus, dihydrofolate reductase requires neither the natural termini nor the cleaved loop for stability, for the conformational changes that accompany catalysis as well as the binding of inhibitors, and for the folding process. 相似文献
4.
Rat mitochondrial DNA polymorphism: sequence analysis of a hypervariable site for insertions/deletions 总被引:7,自引:2,他引:5
下载免费PDF全文
![点击此处可从《Nucleic acids research》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Three closely related variants of rat (Rattus norvegicus) mtDNA have been shown to differ in the number of T residues found in a run of Ts (light strand) which spans the junction between the tRNACys and tRNATyr genes. The number of Ts in the repeat varies from 6 to 8 in these DNAs. Another, less closely related, R. norvegicus variant has a run of 5 Ts at this site and in the related species, Rattus rattus, a run of 4 Ts is found. In R. norvegicus mtDNA runs of 5 As and 5 Gs are found just to the 3' side of the variable T repeat, and it is suggested that the three runs of repeated nucleotides may stabilize heteroduplexes which result from strand slippage and which give rise to the insertions and/or deletions. Among 17 mtDNA clones derived from an individual with the 8T repeat, one clone was found which possessed a 9T repeat. This variant may represent an additional DNA type originally present within the individual. 相似文献
5.
Effects of multiple replacements at a single position on the folding and stability of dihydrofolate reductase from Escherichia coli 总被引:5,自引:0,他引:5
We have made multiple replacements (alanine, arginine, cysteine, histidine, isoleucine, serine, tyrosine) of valine-75 in dihydrofolate reductase from Escherichia coli to examine the relative importance to protein folding of the position that is substituted and the specific character of the amino acid replacement. Valine-75 is part of the eight-stranded beta sheet that forms the structural core of the protein. The isopropyl side chain participates in van der Waals interactions with a number of nonpolar residues, helping to establish a large hydrophobic cluster. Equilibrium studies showed that arginine, histidine, isoleucine, serine, and tyrosine destabilize the protein by 1.9-2.8 kcal mol-1. Alanine and cysteine substitutions have little or no effect. Contrary to other recent studies of the effect of multiple replacements at a hydrophobic site, there is no observed correlation between the changes of the free energy of folding and the changes of the free energy of transfer for the individual amino acids from water to an organic solvent when they are inserted into this site. The effects observed in kinetic studies are both consistent with and extend the equilibrium results; these data indicate that position 75 participates in a rate-limiting step of folding. Some of the equilibrium and kinetic properties of the tyrosine-75 mutant deviated significantly from those of wild-type protein and the other mutants at position 75. (1) The tyrosine variant displayed a complex banding pattern when analyzed by native gel electrophoresis; the wild-type protein and all other mutants at position 75 migrated as single, discrete bands. (2) Comparison of the difference ultraviolet and circular dichroism transition curves showed that a third species is populated at equilibrium; the wild-type protein and all other mutants at position 75 follow a two-state model involving only native and unfolded forms. (3) A third kinetic phase appeared in the unfolding reaction; the wild-type protein and all other mutants at position 75 only showed two kinetic phases in unfolding. Properties 1 and 3 suggest that the tyrosine mutation significantly alters the distribution of native conformers in the protein. These effects on the equilibrium and kinetic data readily display an overriding pattern: residues that would require hydrogen bonding or lead to an expansion of the tightly packed hydrophobic environment in which valine-75 resides destabilize the protein and alter relaxation times of kinetic phases in a consistent manner.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
6.
7.
A protein target site in an early replicated human DNA sequence: a highly conserved binding motif 总被引:2,自引:0,他引:2
M Giacca M I Gutierrez F Demarchi S Diviacco G Biamonti S Riva A Falaschi 《Biochemical and biophysical research communications》1989,165(3):956-965
We have previously reported that a human nuclear factor, probably corresponding to the USF/MLTF protein [1,2], is able to bind specifically to a DNA sequence present in DNA replicated at the onset of S-phase [3]. Here we demonstrate that the same factor binds also to several other similar sequences, present in eukaryotic and viral genomes. Mutations or methylation in a CpG dinucleotide, central in the palindromic binding site, completely abolish binding. Furthermore, we present evidence for the existence of at least two other nuclear proteins in human cells with the same DNA binding specificity. The data presented suggest a strong evolutionary conservation, among distantly related organisms, of the binding motif, which is probably the target of a number of nuclear factors that share the same DNA binding specificity albeit in the context of different functions. 相似文献
8.
Carlo Morandi Jeffrey N. Masters Monica Mottes Giuseppe Attardi 《Journal of molecular biology》1982,156(3):583-607
The programming capacity for the synthesis of human dihydrofolic acid reductase in a rabbit reticulocyte lysate has been found to be greatly enhanced in the polysomal poly(A)-containing RNA from a methotrexate-resistant human cell variant (6A3), as compared to the RNA from its parental line (VA2-B). A major fraction of this promoting activity is associated with a 3.8 × 103 base RNA species detectable as a band in the ethidium bromide-stained electrophoretic pattern of the RNA from 6A3 cells, but not in the RNA from VA2-B cells. Furthermore, sucrose gradient fractionation experiments have indicated that another substantial portion of the messenger activity is associated with RNA components around 103 bases in size. Double-stranded complementary DNA synthesized from total poly(A)-containing RNA of 6A3 cells has been size fractionated, and both large (1400 to 3800 base-pairs) and small size complementary DNA (600 to 1400 base-pairs) species have been used separately to transform Escherichia coli χ2282 with pBR322 as a vector. Of 76 transformants obtained with the large size complementary DNA, identified by a differential colony hybridization assay, none has expressed the dihydrofolic acid reductase coding sequence in E. coli, as judged by resistance to trimethoprim. By contrast, eight trimethoprim-resistant transformants have been obtained using the small size complementary DNA, and their plasmids have been shown to contain the dihydrofolic acid reductase coding sequence by restriction mapping and DNA sequencing; moreover, immunoautoradiographic experiments have revealed the presence in the extracts of two of these transformants of a protein with the electrophoretic mobility and immunoreactivity of human dihydrofolic acid reductase. Restriction mapping and DNA transfer hybridization experiments have further indicated that the inserts of the chimaeric plasmids conferring trimethoprim resistance upon the host and of those lacking this capacity cover together a complementary DNA region of about 3.35 × 103 base-pairs, in which the 564 base-pair dihydrofolic acid reductase coding stretch is located near the 5′ end of the sense strand. RNA transfer hybridization experiments using different cloned complementary DNA fragments as probes have shown the presence of three species of dihydrofolic acid reductase-specific messenger RNAs, with sizes of 3.8 × 103, 1.0 × 103 and 0.8 × 103 bases, differing in the length of the 3′ untranslated region, in the poly(A)-containing RNA from two methotrexate-resistant variants, 6A3 and 10B3, and, in greatly reduced amounts, in the RNA from their respective parents, VA2B and HeLa BU25. 相似文献
9.
10.
11.
12.
R67 dihydrofolate reductase (DHFR) is an enzyme, encoded by an R-plasmid, that confers resistance to the antibacterial agent trimethoprim. This homotetramer possesses a single active site pore and exact 222 symmetry. The symmetry imposes constraints on the ability of the enzyme to optimize binding of the substrate, dihydrofolate (DHF), and the cofactor, NADPH, resulting in a "one site fits both ligands" approach. This approach allows formation of either a NADPH.NADPH, dihydrofolate.dihydrofolate, or NADPH.dihydrofolate complex. The first two complexes are nonproductive, while the third is the productive catalytic species. To break the symmetry of the active site, a tandem array of four R67 DHFR genes has been linked in frame, allowing individual manipulation of each gene copy. Various numbers and combinations of asymmetric Q67H mutations have been engineered into the tandem gene array. The Q67H mutation was chosen for investigation as it was previously found to tighten binding to both dihydrofolate and NADPH by approximately 100-fold in homotetrameric R67 DHFR [Park, H., Bradrick, T. D., and Howell, E. E. (1997) Protein Eng. 10, 1415-1424]. Nonadditive effects on ligand binding are observed when one to four mutations are inserted, indicating either conformational changes in the protein or different cooperativity patterns in the ligand-ligand interactions. From steady state kinetics, addition of Q67H mutations does not drastically affect formation of the NADPH.dihydrofolate complex; however, a large energy difference between the productive and nonproductive complexes is no longer maintained. A role for Q67 in discriminating between these various states is proposed. Since theories of protein evolution suggest gene duplication followed by accumulation of mutations can lead to divergence of activity, this study is a first step toward asking if introduction of asymmetric mutations in the quadrupled R67 DHFR gene can lead to optimization of ligand binding sites. 相似文献
13.
Replication in the amplified dihydrofolate reductase domain in CHO cells may initiate at two distinct sites, one of which is a repetitive sequence element. 总被引:8,自引:22,他引:8
下载免费PDF全文
![点击此处可从《Molecular and cellular biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
To study initiation of DNA replication in mammalian chromosomes, we have established a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) that contains approximately 1,000 copies of the early replicating dihydrofolate reductase (DHFR) domain. We have previously shown that DNA replication in the prevalent 243-kilobase (kb) amplicon type in this cell line initiates somewhere within a 28-kb region located downstream from the DHFR gene. In an attempt to localize the origin of replication with more precision, we blocked the progress of replication forks emanating from origins at the beginning of the S phase by the introduction of trioxsalen cross-links at 1- to 5-kb intervals in the parental double-stranded DNA. The small DNA fragments synthesized under these conditions (which should be centered around replication origins) were then used as hybridization probes on digests of cosmids and plasmids from the DHFR domain. These studies suggested that in cells synchronized by this regimen, DNA replication initiates at two separate sites within the previously defined 28-kb replication initiation locus, in general agreement with results described in the accompanying paper (T.-H. Leu and J. L. Hamlin, Mol. Cell. Biol. 9:523-531, 1989). One of these sites contains a repeated DNA sequence element that is found at or near many other initiation sites in the genome, since it was also highly enriched in the early replicating DNA isolated from cross-linked CHO cells that contain only two copies of the DHFR domain. 相似文献
14.
We describe a new method for obtaining DNA fragments starting at a desired point where there is no recognition sequence for any known restriction endonuclease. A single-stranded DNA containing the fragment of interest is annealed to a synthetic oligonucleotide hybridizing at the 5' end of the required fragment. Then, a partially double-stranded DNA is synthesized using the Klenow fragment of DNA polymerase I in the presence of the four deoxynucleoside triphosphates. The remaining single-stranded regions are removed by digestion with a single-strand nuclease, and the resulting 5' blunt-ended fragment is finally released by digestion with a restriction endonuclease at any site downstream its 3' end. The usefulness of the method was exemplified here by insertion of an epidermal growth factor-like African swine fever virus gene immediately downstream of the ribosome binding site of an expression vector. 相似文献
15.
16.
The human salivary amylase gene (amy1), consisting of eleven exons, is expressed in the salivary gland and in some amylase-producing tumors. Its uppermost exon and the following intron, along with the 5'-flanking region of this gene, are shown to be superimposed with a gamma-actin pseudogene sequence, a portion of which is transcribed into salivary amylase mRNA and another portion of which serves as a promoter for the amy1 gene. In the further upstream region, the gamma-actin pseudogene sequence is interrupted by a human endogenous retroviral nucleotide sequence. 相似文献
17.
Amplification of dsDNA by polymerase chain reaction (PCR) has been limited to those instances in which segments of known sequence flank the fragment to be amplified. A strategy for the PCR amplification of cloned or genomic dsDNA that necessitates sequence information from only a single short segment (single site PCR) has been devised. The region of known sequence may be located at any position within or adjacent to the segment to be amplified. The basic procedure for amplification consists of 1) digestion of dsDNA with one or more restriction enzymes, 2) ligation with a universal anchor adaptor and 3) PCR amplification using an anchor primer and the primer for the single site of known sequence. The anchor adaptor is designed in such a way as to facilitate the amplification of only those fragments containing the sequence of interest. We have demonstrated the utility of this technique by specifically amplifying and directly sequencing antibody variable region genes from cloned dsDNA and from genomic DNA. 相似文献
18.
A mouse genomic clone containing a lactate dehydrogenase-A (LDH-A)
processed pseudogene and a B1 repetitive element was isolated, and a
nucleotide sequence of approximately 3 kb was determined. The pseudogene
and B1 element are flanked by perfect 13-bp repeats, and the B1 sequence
starts at 14 nucleotides 3' to the presumptive polyadenylation signal of
the pseudogene. The nucleotide sequences of the LDH-A genes and processed
pseudogenes from mouse, rat, and human were compared, and a phylogenetic
tree was constructed. The rate and pattern of nucleotide substitutions in
the LDH-A pseudogenes are similar to previously reported results (Li et al.
1984). The average rate of nucleotide substitutions in the LDH-A
pseudogenes is 4.3 X 10(- 9)/site/year. The substitutions of C----T and
G----A are most frequent, and A----G substitutions are relatively high. The
rate of synonymous substitutions in the LDH-A genes is 5.3 X 10(-9), which
is not significantly higher than the average rate of 4.7 X 10(-9) for 35
mammalian genes. The rate of nonsynonymous substitutions in the LDH-A genes
is 0.20 X 10(-9), which is considerably lower than the average rate of 0.88
X 10(-9) for 35 mammalian genes. Thus, the mammalian LDH-A gene appears to
be highly conserved in evolution.
相似文献
19.
Multiple variants of the human lymphocyte homing receptor CD44 generated by insertions at a single site in the extracellular domain. 总被引:17,自引:0,他引:17
The human CD44 cell-surface glycoprotein participates in a wide variety of cell-cell interactions including lymphocyte homing and tumor metastasis. The CD44 antigen is known to display extensive size heterogeneity when compared between different tissue sources although the structural basis for this variation is not yet clear. Recently, two further isotypes in addition to the basic hemopoietic form of the CD44 antigen have been cloned and sequenced and these have been found to contain all or part of a 200-400-base pair insert within the extracellular domain, suggesting that the characteristic heterogeneity in the molecule may be generated by a mechanism of alternative splicing. We have obtained further evidence for alternative splicing, and we report here the cloning and sequencing of six different CD44 sequence variants from a variety of cell lines using a combination of expression cloning and the polymerase chain reaction. Comparison of these variants indicates that each is probably assembled by the insertion of five different exon units in tandem into a discrete site within the membrane proximal region of the extracellular domain. One of the variants contains an exon that shares extensive amino acid sequence homology with a recently described rat CD44 variant that mediates tumor metastasis. Another variant contains a new exon that encodes a tandem repeat of the consensus sequence SG for covalent modification with chondroitin sulfate and is expressed predominantly on mammary tumors. We suggest that a mechanism of alternative exon splicing generates much of the observed structural heterogeneity of CD44 and that the particular set of CD44 variants expressed in a single cell may represent a precise postal code directing the final destination of migrating cells and metastatic tumors. 相似文献
20.
Klon AE Héroux A Ross LJ Pathak V Johnson CA Piper JR Borhani DW 《Journal of molecular biology》2002,320(3):677-693
The crystal structures of two human dihydrofolate reductase (hDHFR) ternary complexes, each with bound NADPH cofactor and a lipophilic antifolate inhibitor, have been determined at atomic resolution. The potent inhibitors 6-([5-quinolylamino]methyl)-2,4-diamino-5-methylpyrido[2,3-d]pyrimidine (SRI-9439) and (Z)-6-(2-[2,5-dimethoxyphenyl]ethen-1-yl)-2,4-diamino-5-methylpyrido[2,3-d]pyrimidine (SRI-9662) were developed at Southern Research Institute against Toxoplasma gondii DHFR-thymidylate synthase. The 5-deazapteridine ring of each inhibitor adopts an unusual puckered conformation that enables the formation of identical contacts in the active site. Conversely, the quinoline and dimethoxybenzene moieties exhibit distinct binding characteristics that account for the differences in inhibitory activity. In both structures, a salt-bridge is formed between Arg70 in the active site and Glu44 from a symmetry-related molecule in the crystal lattice that mimics the binding of methotrexate to DHFR. 相似文献