Sequences In Mammalian Cells Research Articles

Histone modification in constitutive heterochromatin versus unexpressed euchromatin in human cells.

Histone modifications are implicated in regulating chromatin condensation but it is unclear how they differ between constitutive heterochromatin and unexpressed euchromatin. Chromatin immunoprecipitation (ChIP) assays were done on various human cell populations using antibodies specific for acetylated or methylated forms of histone H3 or H4. Analysis of the immunoprecipitates was by quantitative real-time PCR or semi-quantitative PCR (SQ-PCR). Of eight tested antibodies, the one for histone H4 acetylated at lysine 4, 8, 12, or 16 was best for distinguishing constitutive heterochromatin from unexpressed euchromatin, but differences in the extent of immunoprecipitation of these two types of chromatin were only modest, although highly reproducible. With this antibody, there was an average of 2.5-fold less immunoprecipitation of three constitutive heterochromatin regions than of four unexpressed euchromatic gene regions and about 15-fold less immunoprecipitation of these heterochromatin standards than of two constitutively expressed gene standards (P <0.001). We also analyzed histone acetylation and methylation by immunocytochemistry with antibodies to H4 acetylated at lysine 8, H3 trimethylated at lysine 9, and H3 methylated at lysine 4. In addition, immunocytochemical analysis was done with an antibody to heterochromatin protein 1alpha (HP1alpha), whose preferential binding to heterochromatin has been linked to trimethylation of H3 at lysine 9. Our combined ChIP and immunocytochemical results suggest that factors other than hypoacetylation of the N-terminal tails of H4 and hypermethylation of H3 at lysine 9 can play an important role in determining whether a chromatin sequence in mammalian cells is constitutively heterochromatic.

The replicon revisited: an old model learns new tricks in metazoan chromosomes.

The origins of DNA replication were proposed in the replicon model to be specified genetically by replicator elements that coordinate the initiation of DNA synthesis with gene expression and cell growth. Recent studies have identified DNA sequences in mammalian cells that fulfil the genetic criteria for replicators and are beginning to uncover the sequence requirements for the initiation of DNA replication. Mammalian replicators are com- posed of non-redundant modules that cooperate to direct initiation to specific chromosomal sites. Conversely, replicators do not show strong sequence similarity, and their ability to initiate replication depends on the chromosomal context and epigenetic factors, as well as their primary sequence. Here, we review the properties of metazoan replicators, and discuss the genetic and epigenetic factors that determine where and when DNA replication is initiated.

An Effective Method for Selecting siRNA Target Sequences in Mammalian Cells

RNA interference is a gene-silencing phenomenon triggered by dsRNA (double-stranded RNA) and has been widely used for studying gene functions. The short interfering RNA (siRNA) responsible for RNA interference, however, varies markedly in its gene-silencing efficacy. Because this efficacy depends on the selected target sequences, we developed an effective selection method based on the gene degradation measure (priority score) defined by positional features of individual nucleotides. We tested this method experimentally by using it to select new siRNA target sequences in the homo sapiens cyclin B1 gene (CCNB1) and confirmed that it selected highly effective gene-silencing sequences. The proposed method will therefore be useful for selecting new siRNA target sequences in mammalian cells.

The human beta-globin replication initiation region consists of two modular independent replicators.

Previous studies have shown that mammalian cells contain replicator sequences, which can determine where DNA replication initiates. However, the specific sequences that confer replicator activity were not identified. Here we report a detailed analysis of replicator sequences that dictate initiation of DNA replication from the human beta-globin locus. This analysis suggests that the beta-globin replication initiation region contains two adjacent, redundant replicators. Each replicator was capable of initiating DNA replication independently at ectopic sites. Within each of these two replicators, we identified short, discrete, nonredundant sequences, which cooperatively determine replicator activity. Experiments with somatic cell hybrids further demonstrated that the requirements for initiation at ectopic sites were similar to the requirements for initiation within native human chromosomes. The replicator clustering and redundancy exemplified in the human beta-globin locus may account for the extreme difficulty in identifying replicator sequences in mammalian cells and suggest that mammalian replication initiation sites may be determined by cooperative sequence modules.

Chromatin immunoprecipitation.

Fundamental parameters of biological control require sequence-specific protein-deoxyribonucleic acid (DNA) interactions. The packaging of DNA into higher order chromatin controls accessibility to DNA binding proteins that influence regulatory events that include transcription, replication, recombination, and DNA repair. Chromatin is remodeled in a physiologically responsive manner by covalently modifying histones to neutralize a positive charge of these basic chromosomal proteins that form nucleosomes, the primary unit of nucleoprotein structure. Acetylation and methylation of residues on the unstructured tails of histone proteins are the most prevalent chromatin modification related to transcriptional regulation. To understand chromatin structure of a gene, it is important to characterize the interactions of structural and regulatory proteins in the vicinity of covalently modified nucleosomes. Formaldehyde crosslinking rapidly fixes protein-protein and protein-DNA complexes in vivo providing the basis for an approach to analyze native structure of gene promoters. The protocol below is a simple yet sensitive method to determine whether a known protein is associated with a DNA sequence in mammalian cells. It must be noted that this protocol is not limited to sequence-specific DNA binding proteins. The success of this procedure relies on the use of an antibody that specifically and tightly binds its target protein in the buffer, and wash conditions used for the chromatin immunoprecipitation (ChIP) assay. Briefly, the protein-protein and protein-DNA complexes in vivo are reversibly crosslinked by formaldehyde.

Cadherin sequences that inhibit beta-catenin signaling: a study in yeast and mammalian cells.

Drosophila Armadillo and its mammalian homologue beta-catenin are scaffolding proteins involved in the assembly of multiprotein complexes with diverse biological roles. They mediate adherens junction assembly, thus determining tissue architecture, and also transduce Wnt/Wingless intercellular signals, which regulate embryonic cell fates and, if inappropriately activated, contribute to tumorigenesis. To learn more about Armadillo/beta-catenin's scaffolding function, we examined in detail its interaction with one of its protein targets, cadherin. We utilized two assay systems: the yeast two-hybrid system to study cadherin binding in the absence of Armadillo/beta-catenin's other protein partners, and mammalian cells where interactions were assessed in their presence. We found that segments of the cadherin cytoplasmic tail as small as 23 amino acids bind Armadillo or beta-catenin in yeast, whereas a slightly longer region is required for binding in mammalian cells. We used mutagenesis to identify critical amino acids required for cadherin interaction with Armadillo/beta-catenin. Expression of such short cadherin sequences in mammalian cells did not affect adherens junctions but effectively inhibited beta-catenin-mediated signaling. This suggests that the interaction between beta-catenin and T cell factor family transcription factors is a sensitive target for disruption, making the use of analogues of these cadherin derivatives a potentially useful means to suppress tumor progression.

Targeted gene correction by small single-stranded oligonucleotides in mammalian cells.

We demonstrate that relatively short single-stranded oligodeoxynucleotides, 25-61 bases homologous to the target sequence except for a single mismatch to the targeted base, are capable of correcting a single point mutation (G to A) in the mutant beta-galactosidase gene, in nuclear extracts, episome, and chromosome of mammalian cells, with correction rates of approximately 0.05%, 1% and 0.1%, respectively. Surprisingly, these short single-stranded oligonucleotides (ODN) showed a similar gene correction frequency to chimeric RNA-DNA oligonucleotide, measured using the same system. The in vitro gene correction induced by ODN in nuclear extracts was not dependent on the length or polarity of the oligonucleotide. In contrast, the episomal and chromosomal gene corrections were highly dependent on the ODN length and polarity. ODN with a homology of 45 nucleotides showed the highest frequency and ODN with antisense orientation showed a 1000-fold higher frequency than sense orientation, indicating a possible influence of transcription on gene correction. Deoxyoligonucleotides showed a higher frequency of gene correction than ribo-oligonucleotides of the identical sequence. These results show that a relatively short ODN can make a sequence-specific change in the target sequence in mammalian cells, at a similar frequency as the chimeric RNA-DNA oligonucleotide.

Selection of homeotic proteins for binding to a human DNA replication origin

We have previously shown that a cell cycle-dependent nucleoprotein complex assembles in vivo on a 74 bp sequence within the human DNA replication origin associated to the Lamin B2 gene. Here, we report the identification, using a one-hybrid screen in yeast, of three proteins interacting with the 74 bp sequence. All of them, namely HOXA13, HOXC10 and HOXC13, are orthologues of the Abdominal-B gene of Drosophila melanogaster and are members of the homeogene family of developmental regulators. We describe the complete open reading frame sequence of HOXC10 and HOXC13 along with the structure of the HoxC13 gene. The specificity of binding of these two proteins to the Lamin B2 origin is confirmed by both band-shift and in vitro footprinting assays. In addition, the ability of HOXC10 and HOXC13 to increase the activity of a promoter containing the 74 bp sequence, as assayed by CAT-assay experiments, demonstrates a direct interaction of these homeoproteins with the origin sequence in mammalian cells. We also show that HOXC10 expression is cell-type-dependent and positively correlates with cell proliferation.

Intrachromosomal recombination between well-separated, homologous sequences in mammalian cells.

In the present study, we investigated intrachromosomal homologous recombination in a murine hybridoma in which the recipient for recombination, the haploid, endogenous chromosomal immunoglobulin mu-gene bearing a mutation in the constant (Cmu) region, was separated from the integrated single copy wild-type donor Cmu region by approximately 1 Mb along the hybridoma chromosome. Homologous recombination between the donor and recipient Cmu region occurred with high frequency, correcting the mutant chromosomal mu-gene in the hybridoma. This enabled recombinant hybridomas to synthesize normal IgM and to be detected as plaque-forming cells (PFC). Characterization of the recombinants revealed that they could be placed into three distinct classes. The generation of the class I recombinants was consistent with a simple unequal sister chromatid exchange (USCE) between the donor and recipient Cmu region, as they contained the three Cmu-bearing fragments expected from this recombination, the original donor Cmu region along with both products of the single reciprocal crossover. However, a simple mechanism of homologous recombination was not sufficient in explaining the more complex Cmu region structures characterizing the class II and class III recombinants. To explain these recombinants, a model is proposed in which unequal pairing between the donor and recipient Cmu regions located on sister chromatids resulted in two crossover events. One crossover resulted in the deletion of sequences from one chromatid forming a DNA circle, which then integrated into the sister chromatid by a second reciprocal crossover.

Extrachromosomal unequal homologous recombination and gene conversion in simian kidney cells: effects of UV damage

Shuttle plasmid vectors containing the SV40 origin of replication and tandem neo genes with distally placed non-overlapping deletions were used to study the effects of DNA damage on extrachromosomal homologous recombination in simian kidney cells. DNA was introduced into COS7 cells by a lipofectin-mediated transfection procedure and recombination was assessed by analyzing the structure of plasmids. Recombinational events observed included unequal homologous recombination (triplication), gene conversion, double reciprocal recombination, deletion (pop-outs), gene amplification (4–6 copies), and multimerization. Triplication, an event that previously had not been reported in association with extrachromosomal recombination, predominated in experiments with undamaged vectors. The recombination frequency (NeoR/AmpR) of vectors randomly damaged by UV irradiation was essentially unchanged; however, the relative number of triplication events decreased significantly. Selective damage in one of the two neo genes increased the relative frequency of gene conversion. The experimental system developed for use in this study detects all major homologous recombination events observed in chromosomal direct repeat sequences in mammalian cells and yeast and should prove valuable for future studies of homologous recombination in mammalian cells.

Mapping Replication Origins by Quantifying Relative Abundance of Nascent DNA Strands Using Competitive Polymerase Chain Reaction

A procedure was developed for mapping origins of DNA replication in mammalian cell chromosomes based on determining the relative abundance of nascent DNA strands throughout a specific genomic region. The method entails purification of short strands of nascent DNA derived from recently activated origins and the quantification, within this sample, of the relative abundances of different adjacent DNA segments by a competitive polymerase chain reaction technique. It is expected that the abundance of defined markers within the origin region is greatest at the site where DNA replication begins. This origin mapping procedure (i) allows analysis of single-copy genomic regions, (ii) can be performed on cultured and primary cells in the absence of any chemical treatment, (iii) does not require cell synchronization, and (iv) allows mapping origins to within a few hundred base pairs. This high degree of resolution permits a study of thecis- andtrans-acting elements required for origin function. Application of this method to single-copy sequences in mammalian cells has identified replication origins within an ∼500-bp segment in the human lamin B2 gene domain and within an ∼800-bp segment in the hamster dihydrofolate reductase gene locus.

K-ras codon 12 and 61 point mutations in bromodeoxyuridine- and N-nitrosomethylurea-induced rat renal mesenchymal tumors.

The mutagenic thymidine analog bromodeoxyuridine (BrdUrd) may incorrectly incorporate opposite deoxyguanine in DNA, then pair with deoxyadenosine during subsequent replication. It appears to preferentially target the 3'-G of 5'-NGGN-3' sequences in mammalian cells in culture to induce G-->A transitions. Ras genes should therefore be vulnerable to activation by mutation at glycine codons 12 (GGT) and/or 13 (GGC) by misincorporation of BrdUrd. There is limited evidence that BrdUrd may be carcinogenic or co-carcinogenic in rats: three renal mesenchymal tumors, a tumor known to be associated with activating mutations in the c-K-ras-2 oncogene, were reported in 87 rats treated with BrdUrd alone, while N-nitrosomethylurea (NMU) alone or NMU + BrdUrd resulted in incidences of 12/52 and 26/76, respectively, against a zero incidence in untreated rats. We analyzed renal mesenchymal tumors from rats treated with BrdUrd for mutations in K-ras exons 1 and 2 and compared the prevalence and spectrum of mutations with those found in comparable tumors induced with NMU. DNAs from 22 paraffin-embedded renal mesenchymal tumors from rats treated 12-15 months earlier with BrdUrd (three specimens) or NMU (11 specimens) or both agents sequentially (eight specimens) were amplified by PCR. The base sequence of codons 12-13 and 59-63 of K-ras was determined by the dideoxynucleotide method. Sequencing results were confirmed by allele-specific oligonucleotide hybridization. Two of three tumors that appeared in rats given BrdUrd alone contained both a codon 12 GGT-->GAT transition and a codon 61 CAA-->CTA transversion. One tumor induced by NMU alone also showed a codon 12 GGT-->GAT mutation, while only wild type sequence could be demonstrated in the codon 12-13 region in the remaining ten such tumors. Three NMU-induced tumors also showed codon 61 CAA-->CTA mutations, while the remaining tumors had wild type sequence. While the GGT-->GAT transitions identified in tumors from BrdUrd-treated rats are consistent with BrdUrd mutagenesis by misincorporation, the co-occurrence of CAA-->CTA transversions, the overall low prevalence of mutations, and the lack of any difference in mutation spectrum between tumors induced by NMU and those that occurred in BrdUrd-treated rats suggests that in both groups the mutations that did occur did not result from a direct effect of either agent.

Secretion of Active Fc Fragments of Immunoglobulin E Directed by the Yeast Invertase Signal Sequence in Mammalian Cells.

The human Fc fragments of immunoglobulin E (IgE-Fc) expressed in the mammalian COS cells, those mainly consisting of C epsilon 3-C epsilon 4 chains with or without Cys-328 known to be responsible for interchain disulfide bonding, were secreted into the culture medium directed by the yeast Saccharomyces cerevisiae invertase (SUC2) signal sequence (SUC2/Ig2 or SUC2/Ig, respectively) as well as by the human interleukin 2 receptor alpha chain signal sequence (IL2R/Ig2 or IL2R/Ig, respectively). For the binding activity to the human soluble alpha chain of high affinity receptor for IgE, IgE-Fc with Cys328, SUC2/Ig2 and IL2R/Ig2, were active but had smaller activity than native IgE. By comparison IgE-Fc without Cys328, SUC2/Ig and IL2R/Ig, showed lower activity than SUC2/Ig2 and IL2R/Ig2. Immunoprecipitation analysis revealed both SUC2/Ig and SUC2/Ig2 had molecular weights (M(r)s) and degrees of glycosylation similar to IL2R/Ig and IL2R/Ig2, respectively. These results suggested that in mammalian cells the SUC2 signal sequence was functional in directing the heterologous multimeric proteins to the endoplasmic reticulum, resulting in secretion of the active proteins. This finding might show merits on heterologous protein secretion systems.

Mutagenicity of a unique 8-oxoguanine in a human Ha-ras sequence in mammalian cells.

The processing of a unique 8-oxoguanine residue in DNA has been studied in mammalian cells using a single-stranded shuttle vector. A fragment of human Ha-ras carrying the lesion on the first (G1) or the second guanine (G2) of codon 12 was inserted in a shuttle plasmid. Extrachromosomal DNA is replicated in animal cells, extracted and used to transform bacteria to be amplified and individualized. DNA sequencing of bacterial clones showed the mutagenic potency of 8-oxoguanine in vivo to be approximately 4%. The presence of the 8-oxoguanine does not greatly affect survival of the progeny. No significant difference was observed between the mutation frequencies induced by 8-oxoguanine located either at the G1 or G2 position. The majority of the mutations, targeted at the lesion level, are G to T transversions. These base substitutions induced respectively glycine to cysteine (G1) or valine (G2) change in the P21ras protein. These mutations may contribute to activation of the protooncogene, leading to spontaneous tumorigenesis.

Genetic selection of growth-inhibitory sequences in mammalian cells.

To assess the role of mitogenically activated genes in the control of cell proliferation, we have taken a genetic approach based on the premise that blocking the function of an essential gene should lead to growth inhibition. Using a newly developed selection procedure, we isolated growth-inhibitory sequences from a pool of random cDNA fragments of 19 growth-related genes associated with the G0/G1 transition. These sequences encode potential dominant negative variants of c-Fos, JunB, and p44MAPK that may interfere with their growth-related functions. We anticipate that this procedure, which allows for the selection of sequences that cause a growth-inhibition phenotype, may have broad applications in the identification and analysis of genes that regulate cell growth.

Molecular characterization and expression of the Drosophila Ca2+/calmodulin-dependent protein kinase II gene. Identification of four forms of the enzyme generated from a single gene by alternative splicing.

Four cDNA sequences encoding Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) were isolated from a Drosophila adult head cDNA library using rat CaM kinase II alpha and beta cDNA sequences under low stringency hybridization conditions. These cDNA clones encoded polypeptides of 490, 509, 516, and 530 amino acids, which are identical to one another except for amino acid insertions or deletions near the carboxyl-terminal of the putative "link" segment. These polypeptides showed considerable similarity to rat brain CaM kinase II with more than 70% of the amino acids being identical. The Drosophila adult head contains three major species of CaM kinase II with molecular masses of 55, 58, and 60 kDa. These cross-react with anti-rat CaM kinase II antibody. An expression study of the four cDNA sequences in mammalian cells revealed that the polypeptides of 490, 509, and 530 amino acids that had been predicted from the cDNA sequences correspond to the 55-, 58-, and 60-kDa polypeptides found in the head, respectively, and all exhibited enzymatic properties similar to those of rat brain CaM kinase II, including self-regulation. The Drosophila CaM kinase II gene was located in segment 102E-F on the fourth chromosome and consisted of at least 16 exons spanning approximately 20 kilobase pairs. Four forms of the enzyme are generated from a single gene by alternative splicing. Transcripts of CaM kinase II are expressed in great quantities in the central nervous system in the late embryonic stage of development and are more abundant in the head than in the body of the adult fly.

An in vivo assay for measuring the recombination potential between DNA sequences in mammalian cells

Mammalian intermolecular recombination vectors that place the recombination junction within the intron of a selectable marker gene are presented. Many of the previously reported recombination assays require that recombination occur homologously and that they occur within the coding region of the selectable marker. This vector system involves the use of a human thymidine kinase ( tk) minigene and measures the recombination frequency between any chosen DNA sequences, in mammalian thymidine kinase negative cells. The tk minigene is divided into a 5′ vector and a 3′ vector. In the 5′ vector, the DNA sequence of interest is inserted in the proximal portion of tk intron 2. In the 3′ vector, the DNA sequence of interest is inserted in the intron sequence between the proteolipid protein exon 2 and tk exons 3–7. Recombination through the DNA sequences of interest, either homologous or illegitimate, will reconstruct a functional tk minigene. The recombination junction is spliced out of the transcribed mRNA and thymidine kinase positive cells can be selected in hypoxanthine-aminopterin-thymidine medium. We have tested these vectors to measure the recombination potential of two Alu repetitive sequences (BLUR 8 and BLUR 11) against a control DNA sequence. BLUR 8 and BLUR 11 do not seem to recombine at a significantly higher frequency over that of the control DNA sequence. These recombination vectors display similar sensitivity to previous recombination systems, but allow tremendous flexibility in the choice of potentially recombinogenic sequences.

DNA repair mechanisms and their biologic implications in mammalian cells: Report of a NATO advanced research workshop, Fontevraud, France, Oct. 2–7, 1988

Repair of Alkylation Damage in DNA.- Structure-Function Relationships in Alkylation Damage and Repair.- The Roles of Beta- and Delta-Elimination in the Repair of AP Sites in DNA.- Imidazole Ring-Opened Purines: Occurence and Repair in Escherichia coli and Mammalian Cells.- Substrate Specificity of the Formamidopyrimidine-DNA Glycosylase of E. coli: Repair of the Imidazole Ring-Opened Form of N-Hydroxy-2-Amino-Fluorene-Guanine Adducts in DNA.- Investigation of Sequence Specificity in DNA Alkylation and Repair Using Oligodeoxynucleotide Substrates.- Towards a Role for Promutagenic Lesions in Carcinogenesis.- Acquisition of Resistance to Alkylating Agents by Expression of Methyltransferase Gene in Repair-Deficient Human Cells.- The Mer Minus Phenotype, Patient Response to Nitrosoureas, and Protooncogene Activation in Human Glioblastomas.- The Molecular Basis of Alkylating Agent Resistance in Mammalian Cells.- Differential Repair of O4-Methylthymine and O6-Methylguanine in Rat and Hamster Liver.- Formation of 1, N6-Ethenodeoxyadenosine and 3, N4-Ethenodeoxycytidine in DNA from Several Organs of Rats Exposed to Vinyl Chloride.- The Role of O6-Alkylguanine in Cell Killing and Sister Chromatid Exchange Induced by Alkylating Agents.- Inducible Responses to DNA Damage.- Inducible Repair in Mammalian Cells Treated with Alkylating Agents.- An Update of the Mammalian UV Response: Gene Regulation and Induction of a Protective Function.- DNA Repair Related Events.- The Transportation of AP Endonuclease from Cytoplasm to Nucleus in Mammalian Cells.- DNA Damage and Oxygen Species.- Mutagenesis.- Molecular Studies of Mutagenesis using Plasmid Vectors in Xeroderma Pigmentosum Cells.- Molecular Analysis of Mutations at the HPRT Locus: UV Mutation Spectra in Normal and UV-Sensitive V79 Chinese Hamster Cells.- Comparison of the Mutational Specificities Exhibited by BPDE in Escherichia coli and CHO Cells.- Deoxyribonucleotide Pools, DNA Repair and Mutagenesis.- Recombination and Mismatch Repair.- Duplex-Duplex Homologous Recombination Catalysed by a Human Nuclear Extract. Involvement in Double-Strand Break Repair.- Proteins from Yeast and Human Cells Specific for Model Holliday Junctions in DNA.- Characterization of a Strand Transferase Activity from Human Cells.- Mismatch Repair in Mammalian Cells: Approaches to the In Vitro Study of DNA Mismatch Correction Reactions.- Mismatch Repair Patterns in Simian Cells Correlate with the Specificity of a Mismatch Binding Protein Isolated from Simian and HeLa Cells.- Chromatin Proteins and Chromatin Structure in DNA Repair.- DNA Excision Repair at the Nucleosome Level of Chromatin.- Modulation of Activity of Human Chromatin-Associated Endonucleases on Damaged DNA by Nucleosome Structure.- Relationship Between DNA Repair and Transcription in Defined DNA Sequences in Mammalian Cells.- Non-Random Distribution of UV-Induced Repair in Higher-Order Chromatin Loops in Human Cells and its Relationship to Preferential Repair of Active Genes.- Electron Microscopic Visualization of Chromatin Structure at Sites of DNA Excision Repair Following Ultraviolet Irradiation of Cultured Human Fibroblasts.- Poly(ADP-Ribosyl)ation Reactions and Modulation of Chromatin Structure.- Variation of Nuclear ADP-Ribosyl Transferase in Rat Liver Carcinogenesis and in Synchronized HeLa Cells.- DNA Repair Processes in Mutant Cells Deficient in Poly(ADP-Ribose) Synthesis.- Repair Defective Cell Types.- Co-Recessive Inheritance: A Model for Diseases Associated with Defective DNA Repair.- Mammalian DNA Ligases and the Molecular Defect in Bloom's Syndrome.- Detection of Unique Antigenic Lesions in the Uracil DNA Glycosylase from Bloom's Syndrome.- DNA Ligase Activity in Human Cells from Normal Donors and from Patients with Bloom's Syndrome and Fanconi's Anemia.- Processing of Photoinduced Cross-Links and Monoadducts in Human Cell DNA: Genetic and Molecular Features.- Parallels Between Nucleotide Excision Repair in Human Cells and E. coli.- Mechanisms of Resistance to Ionising Radiations: Genetic and Molecular Studies on Ataxia-Telangiectasia and Related Radiation-Sensitive Mutants.- Defective DNA Synthesis and/or Repair in Amyotrophic Lateral Sclerosis.- Genetic Analysis in Trichothiodystrophy Repair Deficient Cells Confirms the Occurrence of Xeroderma Pigmentosum Group D Mutation in Unrelated Patients.- Chinese Hamster Cell Lines Defective in DNA Repair.- DNA Repair Genes and Oncogenes in DNA Repair.- Human DNA Repair and Recombination Genes.- Molecular Genetic Dissection of Mammalian Excision Repair.- The Cloning and Characterization of a Candidate Gene for the Correction of the Xeroderma D Defect.- Characterization of Chinese Hamster Ovary Cell Mutants Hypersensitive to DNA Damaging Agents.- Oncogene Activation in Xeroderma Pigmentosum Skin Tumors.- Anticancer Drugs in DNA Repair.- Heterogeneity in the O6-Alkylguanine DNA Alkyl-Transferase (AGT) Activity of Human Peripheral Blood Lymphocytes (PBL's).- Formation and Repair of Adducts that Lead to Cross-Links in DNA Treated with Chloro-Ethylating Agents.- Induction of Futile DNA Repair Processing by Bifunctional Intercalators.- Participants.- Author Index.

Determinants of translation efficiency of specific mRNAs in mammalian cells.

The means by which the ribosomes of mammalian cells recognize translation initiation sites and the structural features of an mRNA that define its overall translation efficiency are only partially understood. Even so, an effort to engineer the efficient translation of a specific sequence in mammalian cells can benefit from existing information about mechanisms of translation initiation. The purpose of this article is to review briefly our present knowledge of the factors that influence translation efficiency and to suggest some means by which this knowledge can be exploited to influence the translation efficiency of a specific mRNA.

Induction of the Escherichia coli lactose operon selectively increases repair of its transcribed DNA strand

Nucleotide excision repair helps to ameliorate the lethal and mutagenic consequences of DNA damage by removing helix-distorting lesions from cellular genomes. We have previously analysed the removal of ultraviolet-induced cyclobutane pyrimidine dimers from specific DNA sequences in mammalian cells and demonstrated that transcriptionally active genes are preferentially repaired. Additionally, we found that in rodent and human cells only the transcribed strand of the dihydrofolate reductase gene is selectively repaired. Transcription is blocked by pyrimidine dimers in template DNA and the selective removal of these lesions seems to be important for cell survival after irradiation with ultraviolet light. To determine whether this feature of repair is common to prokaryotes and eukaryotes and better to understand its mechanism, we have investigated repair in the two separate DNA strands of the lactose operon of ultraviolet-irradiated Escherichia coli. We find a dramatic difference in the repair of the two strands only when transcription is induced. Most dimers are removed from the transcribed strand of the induced operon within five minutes of irradiation. In the nontranscribed strand, repair is significantly slower and resembles that found in both strands of the uninduced operon. Thus there seems to be a mechanism that couples nucleotide excision repair and transcription.