Bal31 sensitivity of micronuclear sequences homologous to C4A4/G4T4 repeats in Oxytricha nova

The sequence similarity and functional equivalence of telomeres from macronuclear linear DNA molecules in Oxytricha and telomeric sequences of true mitotic/meiotic chromosomes suggest that the (C4A4)n/(G4T4)n sequences found at macronuclear telomeres may also function as micronuclear telomeres in Oxytricha. In this study, radioactively labeled (C4A4)n have been hybridized to micronuclear DNA samples that have been treated with the enzyme Bal31, which has double-stranded exonuclease activity. A time course of digestion shows that approximately 50% of the micronuclear sequences that hybridize to a C4A4 probe disappear with mild digestion by Bal31, suggesting that these sequences are telomeric. The remainder of the hybridizing sequences are not degraded any more rapidly than the total genomic DNA. All of the C4A4/G4T4 sequences that can be detected by hybridization of C4A4 probes to Southern-blotted restriction enzyme digests of micronuclear DNA occur in regions of the genome that are highly resistant to restriction enzyme digestion and show a clustering of sites reminiscent of telomeres in other organisms. We propose that the micronuclear C4A4 hybridizable sequences that are Bal31 resistant may be located near the telomere and within telomere-associated repetitive sequences that are immediately internal to telomeric (Bal31 sensitive) C4A4 hybridizeable sequences.     

Coreceptor tropism can be influenced by amino acid substitutions in the gp41 transmembrane subunit of human immunodeficiency virus type 1 envelope protein

Many studies have demonstrated that the third variable region (V3) of the human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) is a major determinant of coreceptor tropism. Other regions in the surface gp120 subunit of Env can modulate coreceptor tropism in a manner that is not fully understood. In this study, we evaluated the effect of env determinants outside of V3 on coreceptor usage through the analysis of (i) patient-derived env clones that differ in coreceptor tropism, (ii) chimeric env sequences, and (iii) site-directed mutants. The introduction of distinct V3 sequences from CXCR4-using clones into an R5-tropic env backbone conferred the inefficient use of CXCR4 in some but not all cases. Conversely, in many cases, X4- and dual-tropic env backbones containing the V3 sequences of R5-tropic clones retained the ability to use CXCR4, suggesting that sequences outside of the V3 regions of these CXCR4-using clones were responsible for CXCR4 use. The determinants of CXCR4 use in a set of dual-tropic env sequences with V3 sequences identical to those of R5-tropic clones mapped to the gp41 transmembrane (TM) subunit. In one case, a single-amino-acid substitution in the fusion peptide of TM was able to confer CXCR4 use; however, TM substitutions associated with CXCR4 use varied among different env sequences. These results demonstrate that sequences in TM can modulate coreceptor specificity and that env sequences other than that of V3 may facilitate efficient CXCR4-mediated entry. We hypothesize that the latter plays an important role in the transition from CCR5 to CXCR4 coreceptor use.     

Silent mutations in secondary Shine-Dalgarno sequences in the cDNA of human serum amyloid A4 promotes expression of recombinant protein in Escherichia coli.

The serum amyloid A (SAA) superfamily comprises a number of differentially expressed genes with a high degree of homology in mammalian species. SAA4, an apolipoprotein constitutively expressed only in humans and mice, is associated almost entirely with lipoproteins of the high-density range. The presence of SAA4 mRNA and protein in macrophage-derived foam cells of coronary and carotid arteries suggested a specific role of human SAA4 during inflammation including atherosclerosis. Here we underline the importance of ribosome binding site (rbs)-like sequences (also known as Shine-Dalgarno sequences) in the SAA4 cDNA for expression of recombinant SAA4 protein in Escherichia coli. In contrast to rbs sequences coded by the expression vectors, rbs-like sequences in the cDNA of target protein(s) are known to interfere with protein translation via binding to the small 16S ribosome subunit, yielding low or even no expression. Here we show that PCR mutations of two rbs-like sequences in the human SAA4 cDNA promote expression of considerable amounts of recombinant SAA4 in E.coli.     

Analysis of nucleotide sequences of two ligninase cDNAs from a white-rot filamentous fungus, Phanerochaete chrysosporium

An analysis of nucleotide sequences of two types of ligninase cDNAs isolated from the basidiomycete Phanerochaete chrysosporium, designated CLG4 and CLG5, are presented here. The amino acid sequences of the corresponding ligninase proteins, designated LG4 and LG5, respectively, have been deduced from the cDNA sequences. Mature ligninases LG4 and LG5 are preceded by leader sequences containing 28 and 27 amino acids (aa), respectively, and each contains 344 aa residues. The estimated Mrs of mature LG4 and LG5 are 36,540 and 36,607, respectively. Potential N-glycosylation site(s) with the general sequence Asn-X-Thr/Ser are found in both LG4 and LG5. Nucleotide sequence homology between the coding region of CLG4 and CLG5 is 71.5%, whereas the amino acid sequence homology between the two ligninases is 68.5%. The codon usage of ligninases is extremely biased in favor of codons rich in cytosine and guanine. Amino acid sequences of two tryptic peptides of ligninase H8 have exactly matching sequences in ligninase LG5. Also, the sequences of the oligodeoxynucleotide probes, which correspond to the sequences in the tryptic peptides of ligninase H8 and which were used in isolating the ligninase clones from the cDNA library, have exactly matching sequences in CLG5. The experimentally determined N-terminal sequence of purified ligninase H8 is found in the deduced N-terminal amino acid sequence of LG5. These results suggest that CLG5 encodes ligninase H8 and that CLG4 represents a related but different ligninase gene.     

Recombination of the internal direct repeat element DR2 responsible for the fluidity of the a sequence of herpes simplex virus type 1.

A series of herpes simplex virus type 1 derivatives, having a sequences composed of DR1, Ub, (DR2)3-7, DR4t (a truncated form of DR4), and Uc were isolated and examined. The derivative having a sequences with six copies of DR2 generated progeny viruses having a sequences with the same number (six copies) of DR2. Another derivative, having a sequences with three and seven copies of DR2, generated progeny viruses having a sequences with varied numbers (4, 5, 8, and 10 copies) of DR2, besides the original DR2 arrays (three and seven copies). Therefore, the variation in copy number of DR2 was assumed to be caused mainly by recombination between DR2 arrays rather than by slippage within a DR2 array during DNA replication. The presence of DR2-like sequences in internal direct repeat elements of DR4 and DR3.5 supported the hypothesis of the recombinogenic property of DR2. The equal distribution of divergence of a sequences to both ends of the virus genome favors the double-strand break and gap repair model to explain gene conversion and amplification of the a sequence.     

Preferential DNA repair of (6-4) photoproducts in the dihydrofolate reductase gene of Chinese hamster ovary cells

We have developed a method to quantify (6-4) photoproducts in genes and other specific sequences within the genome. This approach utilizes the following two enzymes from Escherichia coli: ABC excinuclease, a versatile DNA repair enzyme which recognizes many types of lesions in DNA, and DNA photolyase, which reverts pyrimidine dimers. DNA is isolated from UV irradiated Chinese hamster ovary cells and digested with a restriction enzyme. Pyrimidine dimers, the major photoproduct produced at biological UV fluences, are then completely repaired by treatment with DNA photolyase. The photoreactivated DNA is treated with ABC excinuclease, electrophoresed in an alkaline agarose gel, transferred to a support membrane and probed for specific genomic sequences. Net incisions produced by ABC excinuclease following photoreactivation are largely due to the presence of (6-4) photoproducts. These adducts are quantitated by measuring the reduction of intensity of the full length fragments on the autoradiogram. Using this approach we have shown that (6-4) photoproducts are produced at equal frequency in the dihydrofolate reductase coding sequence and in its 3'-flanking, noncoding sequences and that the formation of (6-4) photoproducts is linear in both sequences up to a UV dose of 60 J/m2. The repair of (6-4) photoproducts in these DNA sequences was measured after a dose of 40 J/m2 over 4-, 8-, and 24-h time periods. The (6-4) photoproducts are repaired more efficiently than pyrimidine dimers in both sequences and there is preferential repair of (6-4) photoproducts in the dihydrofolate reductase gene compared with the downstream, noncoding sequences.     

Joint Distribution of Insertion Elements Is4 and Is 5 in Natural Isolates of ESCHERICHIA COLI

A reference collection of natural isolates of Escherichia coli has been studied in order to determine the distribution, abundance and joint occurrence of DNA insertion elements IS4 and IS5. Among these isolates, 36% were found to contain IS4 and 30% were found to contain IS5. Among strains containing IS4 the mean number of copies per strain was 4.4 +/- 0.8; the comparable figure for IS5 was 3.7 +/- 1.0. Although the presence of the elements among the isolates was independent, among those isolates containing both IS4 and IS5, there was a significant negative correlation in the number of copies of the elements. The reference collection was also studied for the presence of the DNA sequences flanking the single copy of IS4 in the chromosome of E. coli K12. Homologous sequences were found in only 26% of the isolates. The sequences flanking the IS4 invariably occur together, and their presence is significantly correlated with the presence of IS4. In eight of the strains that carry these flanking sequences, an IS4 is located between them, and the sequences are present at the homologous position as in the K12 strain. We suggest that IS4 and its flanking sequences share a common mechanism of dissemination, such as plasmids, and we present evidence that they are included in a much larger transposable element.     

Tetraplex folding of telomere sequences and the inclusion of adenine bases.

Telomeres are required for eukaryotic chromosome stability. They consist of regularly repeating guanine-rich sequences, with a single-stranded 3' terminus. Such sequences have been demonstrated to have the propensity to adopt four-stranded structures based on a tetrad of guanine bases. The formation of an intramolecular foldback tetraplex is associated with markedly increased mobility in polyacrylamide. Most telomeric sequences are based either on a repeat of d(TnGGGG) or d(TnAGGG) sequences. We have used a combination 7-deazaguanine or 7-deaza-adenine substitution, chemical modification and gel electrophoresis to address the following aspects of intramolecular tetraplex formation. (i) Intramolecular tetraplex formation by d(TTTTGGGG)4 sequences is prevented by very low levels of 7-deazaguanine substitution. This confirms the important role of guanine N7 in the formation of the tetraplex. (ii) The sequences d(TTAGGG)4 and d(TTTTAGGG)4 fold into tetraplexes. By contrast, the electrophoretic behaviour of d(TTTTGGGA)4, d(TTTTAGAG)4 and d(TTTTGAGA)4 does not indicate formation of stable intramolecular tetraplexes under available conditions. (iii) Selective 7-deazaguanine and 7-deaza-adenine substitutions in d(TTTTAGGG)4 give results consistent with tetraplex folding by the formation of three G4 tetrads, with the adenine bases formally part of the single-stranded loops, where they probably interact with thymine bases. These results demonstrate that eukaryotic cells appear to have selected just those sequences that can adopt the tetraplex conformation for their telomeres, while those that cannot have been avoided. This suggests that the conformation may be significant in the function of the telomere, such as attachment to nuclear structures.     

Characterization of a maize chromosome 4 centromeric sequence: evidence for an evolutionary relationship with the B chromosome centromere

Previous work has identified sequences specific to the B chromosome that are a major component of the B centromere. To address the issue of the origin of the B and the evolution of centromere-localized sequences, DNA prepared from plants without B chromosomes was probed to seek evidence for related sequences. Clones were isolated from maize line B73 without B chromosomes by screening DNA at reduced stringency with a B centromeric probe. These clones were localized to maize centromere 4 using fluorescence in situ hybridization. They showed homology to a maize centromere-mapped sequence, to maize B chromosome centromere sequences, and to a portion of the unit repeat of knobs, which act as neocentromeres in maize. A representative copy was used to screen a BAC library to obtain these sequences in a larger context. Each of the six positive BACs obtained was analyzed to determine the nature of centromere 4-specific sequences present. Fifteen subclones of one BAC were sequenced and the organization of this chromosome 4-specific repeat was examined.     

The structural basis of the multiple forms of human complement component C4

cDNA clones of human complement components C4A and C4B alleles were prepared from mRNA obtained from the liver of a donor heterozygous at both loci. cDNA from one C4A allele was sequenced to give the derived complete amino acid sequence of 1722 amino acid residues of the C4 single chain precursor molecule and the estimated sequences of the three peptide chains of secreted C4. Comparison with partial sequences of a second C4A allele and a C4B allele has led to the tentative identification of some class differences in nucleotide sequences between C4A and C4B and of allelic differences between C4A alleles in this highly polymorphic system.     

Mouse satellite DNA isolated by Ag+ -- CsSO4 density gradients contains G+C rich, slow reassociating sequences

Mouse satellite DNA sequences isolated by centrifugation in CS2SO4--Ag+ gradients are analyzed for buoyant density by CSCl density gradients and for their content of fast reassociating sequences by denaturation and partial reassociation. Our data suggest that in CS2SO4 gradients silver ions separate a satellite band which contains both fast reassociating G+C rich sequences and slow reassociating, A+T rich DNA sequences.     

Internal micronuclear DNA regions which include sequences homologous to macronuclear telomeres are deleted during development in Tetrahymena

C4A2 repeats are present in multiple clusters in both the macronucleus and micronucleus of Tetrahymena. Although the macronucleus is generated from the micronucleus after sexual conjugation, the repeats are telomeric sequences in the macronucleus but are internally located in the micronucleus (1). This study investigates the fate of the sequences adjacent to the micronuclear C4A2 repeats. Southern blot analyses of 21 C4A2-containing micronuclear clones show that extensive elimination of the adjacent sequences occurs during the formation of the macronucleus. Comparison of one C4A2-containing micronuclear clone with its derived macronuclear segment indicates that approximately 4.5 kb of DNA, which includes the C4A2 repeats and adjacent sequences on both sides is deleted from the macronucleus. The two regions adjoining the deletion are joined together to form a contiguous segment in the macronucleus. This excision of C4A2 repeats and surrounding sequences and the rejoining of the retained segments is probably the mechanism by which all or most of the other C4A2 adjacent sequences are eliminated.     

Genomic polymorphism in the T-even bacteriophages.

We have compared the genomes of 49 bacteriophages related to T4. PCR analysis of six chromosomal regions reveals two types of local sequence variation. In four loci, we found only two alternative configurations in all the genomes that could be analyzed. In contrast, two highly polymorphic loci exhibit variations in the number, the order and the identity of the sequences present. In phage T4, both highly polymorphic loci encode internal proteins (IPs) that are encapsidated in the phage particle and injected with the viral DNA. Among the various T4-related phages, 10 different ORFs have been identified in the IP loci; their amino acid sequences have the characteristics of internal proteins. At the beginning of each of these coding sequences is a highly conserved 11 amino acid leader motif. In addition, both 5' and 3' to most of these ORFs, there is a approximately 70 bp sequence that contains a T4 early promoter sequence with an overlapping inversely repeated sequence. The homologies within these flanking sequences may mediate the recombinational shuffling of the IP sequences within the locus. A role for the new IP-like sequences in determining the phage host range is proposed since such a role has been previously demonstrated for the IP1 gene of T4.     

The capsid of the T4 phage superfamily: the evolution, diversity, and structure of some of the most prevalent proteins in the biosphere

The Escherichia coli bacteriophage T4 has served as a classic system in phage biology for more than 60 years. Only recently have phylogenetic analyses and genomic comparisons demonstrated the existence of a large, diverse, and widespread superfamily of T4-like phages in the environment. We report here on the T4-like major capsid protein (MCP) sequences that were obtained by targeted polymerase chain reaction (PCR) of marine environmental samples. This analysis was then expanded to include 1,000 s of new sequences of T4-like capsid genes from the metagenomic data obtained during the Sorcerer II Global Ocean Sampling (GOS) expedition. This data compilation reveals that the diversity of the major and minor capsid proteins from the GOS metagenome follows the same general patterns as the sequences from cultured phage genomes. Interestingly, the new MCP sequences obtained by PCR targeted to MCP sequences in environmental samples are more divergent (deeper branching) than the vast majority of the MCP sequences coming from the other sources. The marine T4-like phage population appears to be largely dominated by the T4-like cyanophages. Using approximately 1,400 T4-like MCP sequences from various sources, we mapped the degree of sequence conservation on a structural model of the T4-like MCP. The results indicate that within the T4 superfamily there are some clear phylogenetic groups with regard to the more conserved and more variable domains of the MCP. Such differences can be correlated with variations in capsid morphology, the arrangement of the MCP lattice, and the presence of different capsid accessory proteins between the subgroups of the T4 superfamily.     

Mouse mammary tumor virus-related sequences in mouse lymphocytes are inducible by 12-O-tetradecanoyl phorbol-13-acetate

cDNA libraries from EL-4 cells treated with 12-O-tetradecanoyl phorbol-13-acetate (TPA) were screened for TPA-inducible sequences by differential hybridization. The most abundant inducible species was a sequence similar to that of mouse mammary tumor virus (MMTV). Induction of the mRNA corresponding to the MMTV-related sequences was already evident 30 min after TPA treatment, whereas the maximum accumulation occurred after 20 h of exposure to TPA. TPA also increased levels of MMTV-related RNA in the normal spleen cells of BALB/c and C57BL/6 mice. The level of RNA expression corresponding to MMTV-related sequences, however, was markedly elevated in EL-4 cells as compared with normal spleen cells. Southern blots of EL-4 cell DNA showed that the MMTV-related sequences were inserted into multiple locations of the EL-4 genome. Sequence analysis revealed that the MMTV-related cDNA clones included a part of the env gene and the right long terminal repeat of MMTV. However, the cDNA sequences were substantially different from published MMTV proviral sequences, most notably because of a contiguous deletion of 491 base pairs in the open reading frame within the U3 region.     

Concerted deletions and inversions are caused by mitotic recombination between delta sequences in Saccharomyces cerevisiae.

Deletions of a tyrosine tRNA suppressor gene, SUP4-o, are mediated by recombination between short repeated delta sequences in Saccharomyces cerevisiae. The arrangement of the five solo delta sequences that surround the SUP4 locus was established by DNA sequence analysis. Seven deletion classes were identified by genomic blotting. DNA sequence analysis also showed that the delta sequences within a 6.5-kilobase region of the SUP4 locus were the endpoints of these events. In three of these classes, an adjacent interval surrounded by delta sequences was inverted in concert with the deletion. The frequency of all deletion classes decreased in strains that contained mutations in the recombination and repair gene RAD52. We present two gene conversion mechanisms by which these rearrangements could have been generated. These models may also explain deletions between repeated sequences in other systems.