Tandem Repeat Structure Research Articles

Isolation and characterization of disulfide-bonded peptides from the three globular domains of aggregating cartilage proteoglycan.

The aggregating cartilage proteoglycan core protein contains two globular domains near the N terminus (G1 and G2) and one near the C terminus (G3). The G1-G3 domains contain 10, 8, and 10 cysteine residues, respectively. The disulfide assignments of the G1 domain have previously been deduced (Neame, P. J., Christner, J. E., and Baker, J. R. (1987) J. Biol. Chem. 262, 17768-17778) as Cys1-Cys2, Cys3-Cys6, Cys4-Cys5, Cys7-Cys10, and Cys8-Cys9, in which the numbers cited after the half-cystine residues are their relative positions from the N terminus. Here we describe a method for the isolation of disulfide-bonded peptides from tryptic digests of bovine nasal cartilage monomer. Sequence analysis of these peptides has allowed us to confirm the pairings previously determined for the G1 domain and to assign a disulfide pattern for the G2 domain of Cys11-Cys14, Cys12-Cys13, Cys15-Cys18, and Cys16-Cys17, in which the Cys15-Cys18 pairing was deduced indirectly. Similarly, for the G3 domain, a pattern of Cys19-Cys20, Cys21-Cys24, Cys22-Cys23, Cys25-Cys27, and Cys26-Cys28 was assigned, in which the Cys22-Cys23 pair was deduced indirectly. The G2 domain therefore contains disulfide bonding which is characteristic of the tandem repeat structures found in the G1 domain and link protein, and the G3 domain contains the three disulfide linkages previously assigned to the family of C-type animal lectins. The method described here, which combines anion-exchange, cation-exchange, and reversed-phase chromatography, should have broad application to the isolation of disulfide-bonded peptides from other heavily glycosylated proteins and proteoglycans.

Development of transgenic mice containing woodchuck hepatitis virus DNA

We produced transgenic mice containing woodchuck hepatitis virus DNA with tandem repeat structure capable of producing virus and viral antigens. Poly(A)+ RNAs probably corresponding to pregenome and viral antigens were detected in their liver. These mice have now been healthy for one year. However, there is the possibility of inducing immunologically mediated hepatitis. We anticipate these transgenic mice may present a useful model system for studying the significance of chronic hepatitis in hepatocellular carcinoma development.

Immunoglobulin fold and tandem repeat structures in proteoglycan N-terminal domains and link protein

Detailed primary sequence and secondary structure analyses are reported for the hyaluronate binding region (G1 domain) and link protein of proteoglycan aggregates † † The nucleotide sequences reported in this paper have been submitted to the GenBank/EMBL Data Bank with accession numbers Y00165 (porcine) and Y00166 (human). . These are based on six full or partial sequences from the chicken, pig, human, rat and bovine proteins. Determinations of a full pig and a partial human link protein sequence are reported in the Appendix. Five sequences at the N terminus in both proteins were compared with the structures of 11 variable immunoglobulin (Ig) fold domains for which crystal structures are available. Despite only modest sequence homology, a clear alignment could be proposed. Analysis of this shows that the equivalents of the first and second hypervariable segments are now significantly longer, and both proteins have N-terminal extensions that are up to 23 residues in length. Secondary structure predictions showed that these sequences could be identified with available crystal structures for the variable Ig fold. However the hydrophobic residues involved in interactions between the light and heavy chains in Igs are replaced by hydrophilic charged groups in both proteins. These results imply that both proteins are members of the Ig superfamily, but exhibit structural differences distinct from other members of this superfamily for which crystal structures are known. The proteoglycan tandem repeat (PTR) is a repeat of 99 residues that is found twice in the amino acid sequence of link protein and the proteoglycan G1 domain adjacent to the Ig fold, and also twice in the proteoglycan G2 domain. A total of 16 PTRs was available for analysis. Compositional analyses show that these are positively charged if these originate from link protein, and negatively charged if from the G1 or G2 domains. The 16 Robson secondary structure predictions for the PTRs were averaged to improve the statistics of the prediction, and checked by comparison with Chou-Fasman calculations. A strong α-helix prediction was found at residues 13 to 25, and several β-strands were predicted. The overall content is 18% α-helix and 28% β-sheet, with 44% of the remaining sequence being predicted as turns. These analyses show that both the proteoglycan G1 domain and link protein are constructed from two distinct globular components, which may provide the two functional roles of these proteins in proteoglycan aggregation.

Isolation and characterization of a novel type of sialoglycoproteins (hyosophorin) from the eggs of medaka, Oryzias latipes: Nonapeptide with a large N-linked glycan chain as a tandem repeat unit

We found a novel type of sialoglycoprotein (SGP) with apparent molecular mass ranging from 15,000 to 100,000 Da in the unfertilized eggs of the medaka fish, Oryzias latipes. From fertilized eggs we isolated the corresponding sialoglycopeptides of apparent molecular weight 7000. The amino acid and carbohydrate compositions of these glycoproteins and glycopeptides are very similar, if not identical, and they contain 90%, by weight, of carbohydrate, the predominant sugars being Gal, GlcNAc, and NeuAc. The chemical and physical data indicate that 15- to 100-kDa SGPs are made up of tandem repeat structures whose repeating unit is 7-kDa sialoglycopeptide, and, upon fertilization, higher molecular weight SGPs undergo proteolytic depolymerization to the least structural unit, 7-kDa sialoglycopeptide. As is the case with polysialoglycoproteins (PSGP) found in salmonid fish eggs, a novel family of sialoglycoproteins has been proven to be a major component of cortical alveoli of medaka eggs, namely, hyosophorin. However, we found that they differ markedly from PSGPs (salmonid fish egg hyosophorins) in terms of the carbohydrate composition. The chemical composition and the results of Smith degradation indicate that SGP contains one large N-linked glycan chain per repeat unit. We have determined the amino acid sequence of 7-kDa sialoglycopeptide: Asp-Ala-Ala-Ser-Asn∗-Gln-Thr-Val-Ser, where ∗ indicates the asparagine residue to which a large glycan chain consisting of Fuc 2Man 3Gal 15GlcNAc 9NeuAc 6 is attached. The direct experimental evidence for the presence of a polyprotein structure suggests that the covalent nature of the higher molecular weight SGPs should be expressed as [Asp-Ala-Ala-Ser-Asn∗-Gln-Thr-Val-Ser] N , where N = 2 to 14 but for the major fraction N = 12.

Alterations in the U3 region of the long terminal repeat of an infectious thymotropic type B retrovirus

We isolated and characterized a type B thymotropic retrovirus (DMBA-LV) which is highly related to mouse mammary tumor virus (MMTV) isolates and which induces T-cell thymomas with a high incidence and a very short latent period. Regions of nonhomology between the DMBA-LV genome and the MMTV genome were identified by heteroduplex mapping and nucleotide sequence studies. In the electron microscope heteroduplex mapping studies the EcoRI-generated 5' and 3' fragments of the DMBA-LV genome were compared with the corresponding fragments of the MMTV (C3H and GR) genome isolated from mammary tumors. The results indicated that DMBA-LV contained a region of nonhomologous nucleotide sequences in the 3' half of the U3 region of the long terminal repeat (LTR). Nucleotide sequence studies confirmed these results and showed that in this region 440 nucleotides of the MMTV (C3H) sequences were deleted and substituted with a segment of 122 nucleotides. This substituted segment in the form of a tandem repeat structure contained nucleotide sequences derived exclusively from sequences which flanked the substitution loop. The distal glucocorticoid regulatory element was unaltered, and two additional copies of the distal glucocorticoid regulatory element-binding site were present in the substituted region. The restriction endonuclease map of the reconstructed molecular clone of DMBA-LV was identical to that corresponding to unintegrated linear DMBA-LV DNA present in DMBA-LV-induced tumor cell lines. Since the nucleotide sequences of the LTRs present in four different DMBA-LV proviral copies isolated from a single thymoma were identical, we concluded that they were derived from the same parental virus and that this type B retrovirus containing an alteration in the U3 region of its LTR could induce thymic lymphomas. Thus, DMBA-LV represents the first example of a productively replicating type B retrovirus that contains an LTR modified in the U3 region and that has target cell and disease specificity for T cells.

Genetic studies on the beta subunit of Escherichia coli RNA polymerase. VIII. Localisation of a region involved in promoter selectivity.

We have previously isolated an E. coli derivative carrying a small internal deletion (delta(rpoB)1570-1) of the beta structural gene. This RNA polymerase deletion mutant has no noticeable phenotype other than a slightly increased generation time in minimal medium. The deletion, which removes about 165 bp, has been localised to between codons 965 and 1,083, indicating it excises part of a tandem repeat structure present in the C-terminal region of beta. Analysis in vitro of purified RNA polymerase from the deletion mutant indicates that this enzyme has an altered promoter selectivity. These observations allow localisation of a site on the beta polypeptide of E. coli RNA polymerase involved in transcriptional initiation.

Transcription of two classes of rat growth hormone gene-associated repetitive DNA: differences in activity and effects of tandem repeat structure.

The rat growth hormone (rGH) gene contains two classes of repetitive DNA arranged as clusters within intron B and the 3' flanking region. The major family is equivalent to the CHO type 2 DNA. The second ("truncated repeat", TR) is a truncated version of the first and occurs in certain neural-specific transcripts and genes ("identifier" elements, ID). Here we report, using the HeLa cell-free transcription assay, that RNA polymerase III (Pol III) efficiently initiates at internal promoters within a tandem array of rGH gene repetitive DNA monomers and results in a novel organization of overlapping Class III transcription units. Transcription competition studies revealed that the rat type 2 structures share Pol III transcription factors with a tRNA gene, a human Alu repeat, and a mutant VA1 gene. Also, the rGH type 2 but not the TR DNA efficiently promotes Pol III initiation, yet other TR members, which differ only in flanking DNA, are transcribed. Thus, the rGH gene is strikingly enriched with 10 repetitive DNA monomers; multimeric type 2 elements are actively transcribed; rGH-TR sequences are expressed only as part of larger transcripts promoted by type 2 DNA; and, type 2 DNA uses tRNA gene transcription factors. These studies show that flanking sequences, promoter organization and factor competition may all affect rat repetitive DNA expression.

Organization of the Epstein-Barr virus DNA molecule. III. Location of the P3HR-1 deletion junction and characterization of the NotI repeat units that form part of the template for an abundant 12-O-tetradecanoylphorbol-13-acetate-induced mRNA transcript.

A 1,400-base-pair (bp) region within the BamHI H fragment of Epstein-Barr virus (EBV) (B95-8) DNA consists of a cluster of tandemly duplicated direct repetitions characterized by single sites for the NotI restriction endonuclease. Nucleotide sequencing revealed a 125-bp repeat unit of 84% guanine-plus-cytosine content which is theoretically capable of extensive secondary structure formation. The flanking sequences adjacent to the NotI repeat cluster contained an additional 38-bp portion of repeat unit DNA, thus establishing that EBV(B95-8) contains a nonintegral number of NotI repeats totalling 11.3 copies. Restriction site mapping of the homologous cloned BamHI "H" fragment from the nontransforming EBV (P3HR-1) isolate revealed that a contiguous 6,650-bp region including the entire NotI repeat cluster has been deleted from the BamHI-H, -Y, and -W regions of the P3HR-1 genome. By nucleotide sequencing across the novel junction, we have precisely identified the P3HR-1 deletion boundaries in BamHI-H and the internal repeat and suggest that a complex pattern of direct and inverted partial DNA homologies may have been involved in the original recombination event. The cloned BamHI H fragment and isolated NotI repeat unit have also been used as probes to detect homologous mRNA transcripts in the B95-8 and Raji cell lines of EBV-transformed lymphoblasts. These experiments showed that the NotI repeats form part of the template for a polyadenylated 2.5-kilobase mRNA transcript which becomes much more abundant after 12-O-tetradecanoyl-phorbol-13-acetate treatment of the cultures. The direction of transcription of this mRNA and the nucleotide sequence of most of its template and 5' flanking regions have been determined. The probable promoter for the 2.5-kilobase mRNA initiates transcription efficiently in an in vitro assay and contains several TAATGA-like elements that may be indicative of herpesvirus immediate-early class promoters. The need to repress expression of this gene during latency suggests a possible novel regulatory role for tandem repeat structures inside a eucaryotic virus transcription unit. The deletion in EBV(P3HR-1), which has been associated with the loss of lymphocyte "immortalizing" capacity in this isolate, eliminates part of the coding region as well as the NotI repeats from the 2.5-kilobase mRNA transcript, but the promoter and proximal 340-bp portions of the template remain.