Hajo Delius

Isolation and characterization of gene 5 protein of filamentous bacterial viruses

Abstract The product of gene 5 of filamentous bacteriophages is required for synthesis of the single-stranded progeny DNA. When extracts made from Escherichia coli infected with bacteriophages fd or M13 are chromatographed on single-stranded DNA/cellulose, a DNA-binding protein can be eluted which has a molecular weight of about 10,000 daltons and is made in at least 100,000 copies per cell. This protein is altered in amber mutant 5-H3 and temperature-sensitive mutant 5-HS1 of phage M13, which identifies it as the gene 5 product (Henry & Pratt, 1969). As judged by sucrose-gradient sedimentation at 4 °C, the pure protein binds tightly and co-operatively to single-stranded, but not to double-stranded, DNAs; at saturation, one protein monomer is bound per every 4 DNA nucleotides. This selective DNA-binding enables the gene 5 protein to denature double-stranded DNAs rapidly at physiological temperatures. In these respects, the gene 5 protein resembles the T4 bacteriophage gene 32 protein described previously, a protein which is required for both the replication and recombination of T4 bacteriophage DNA. However, electron microscopy reveals that the structure of the gene 5 protein complex with single-stranded DNA is quite different: whereas gene 32 protein forces the DNA into an extended linear conformation, the gene 5 protein coalesces two protein-covered DNA stands into a helical, rodlike structure. For this and other reasons, these two “DNA unwinding” proteins could have quite different roles in the replication process.

Dickkopf genes are co-ordinately expressed in mesodermal lineages.

Dickkopf-1 (dkk-1) is member of a novel family of secreted proteins and functions in head induction during Xenopus embryogenesis, acting as a potent inhibitor of Wnt signalling. Here we report: (1) the isolation of two additional murine members of the dkk family, dkk-2 and dkk-3; and (2) analysis of adult and embryonic gene expression of mouse dkk-1,-2, and -3, Xenopus dkk-1 as well as chicken dkk-3. Comparative developmental analyses of the dkk-1, dkk-2 and dkk-3 in mice indicate that these genes are both temporally and spatially regulated. They define overlapping deep domains in mesenchymal lineages suggesting a co-ordinated mode of action. All dkks show distinct and elevated expression patterns in tissues that mediate epithelial- mesenchyme transformations suggesting that they may participate in heart, tooth, hair and whisker follicle, limb and bone induction. In the limb buds expression of these genes are found in regions of programmed cell death. In a given organ, dkk-1 tends to be the earliest member expressed. Comparison with Xenopus dkk-1 and chicken dkk-3 shows evolutionarily conserved expression patterns. Our observations indicate that dkk genes constitute a new family of secreted proteins that may mediate inductive interactions between epithelial and mesenchymal cells.

Mutual antagonism between dickkopf1 and dickkopf2 regulates Wnt/β-catenin signalling

Wnts are secreted glycoproteins implicated in diverse processes during embryonic patterning in metazoans. They signal through seven-transmembrane receptors of the Frizzled (Fz) family [1] to stabilise beta-catenin [2]. Wnts are antagonised by several extracellular inhibitors including the product of the dickkopf1 (dkk1) gene, which was identified in Xenopus embryos and is a member of a multigene family. The dkk1 gene acts upstream of the Wnt pathway component dishevelled but its mechanism of action is unknown [3]. Although the function of Dkk1 as a Wnt inhibitor in vertebrates is well established [3-6], the effect of other Dkks on the Wnt/beta-catenin pathway is unclear. Here, we report that a related family member, Dkk2, activates rather than inhibits the Wnt/beta-catenin signalling pathway in Xenopus embryos. Dkk2 strongly synergised with Wnt receptors of the Fz family to induce Wnt signalling responses. The study identifies Dkk2 as a secreted molecule that is able to activate Wnt/beta-catenin signalling. The results suggest that a coordinated interplay between inhibiting dkk1 and activating dkk2 can modulate Fz signalling.

Characterization by electron microscopy of the complex formed between T4 bacteriophage gene 32-protein and DNA☆

Abstract Techniques have been developed for visualization of gene 32-protein/DNA complexes by electron microscopy. With DNA in excess, gene 32-protein can be seen to bind co-operatively to single-stranded DNA. With protein in excess, all single-strands appear to be uniformly coated with protein. The saturated complex has a flexible rod-like conformation, with a diameter of about 60 A and a length of about 4.6 A per nucleotide. This structure is unchanged in the presence of 1 m m -spermine, a condition which induces extensive folding in the free single-strands. Using glutaraldehyde fixation, gene 32-protein induced denaturation of double-stranded DNAs can be visualized at physiological temperatures. The denaturation map derived with bacteriophage lambda DNA closely resembles that obtained earlier with heat and alkaline denaturation in the absence of protein (Inman, 1967) , indicating that the protein preferentially invades A + T rich double-helical regions, without a requirement for a physical discontinuity such as a cleaved phosphodiester bond or a double-stranded end. With supercoiled DNA, only a single short region of double-helix is opened by gene 32-protein, reflecting the difficulty of denaturation where unwinding is geometrically restricted.

A putative transforming gene of Jijoye virus differs from that of Epstein-Barr virus prototypes

The P3HR-1 strain of Epstein-Barr virus (EBV), a nontransforming clonal derivative of Jijoye (EBV), is characterized by a deletion of 6.6 kb involving part of the BamHI-W repeats and the adjacent region including the NotI repeats. In the transforming parental Jijoye virus this region differs from the corresponding regions in B95-8 or M-ABA virus. The HindIII-B fragments which carry this region from both Jijoye and prototype M-ABA (EBV) viruses have been cloned and subclones have been constructed which contain the left-hand part of HindIII-B from the HindIII to the BglII site (BglII-delta C fragment). By restriction enzyme analysis the inserts were found to be of equal size (6.3 kb) but to differ in their restriction enzyme pattern. Heteroduplexes formed under stringent conditions in the presence of T4 gene 32 protein revealed a substitution loop of 1750 +/- 200 nucleotides. Heteroduplex formation under nonstringent conditions showed that the substituted sequences are partially homologous to each other, with the regions of nonhomology confined to three distinct areas of 100 to 200 nucleotides. The partial homology observed between both regions indicates that they have evolved from a common ancestor. By hybridization of a Jijoye virus subclone containing only sequences of the substituted region to Northern blots a 2.8-kb polyadenylated transcript was detected indicating that the substituted region is expressed in Jijoye cells.

A physical map of wheat chloroplast DNA showing the location of the structural genes for the ribosomal RNAs and the large subunit of ribulose 1,5-bisphosphate carboxylase

SummaryThe restriction endonucleases SalGI and PstI have been used to construct a physical map of wheat ctDNA. The molecule was found to contain approximately 135 kbq, and in common with many other higher plant ctDNAs about 15% of the sequences are repeated in an inverted orientation. It was established by electron microscopy that, in wheat, each segment of the inverted repeat contains 21.0 kbp, and that the single copy regions separating the two repeated segments contain 12.8 kbp and 80.2 kbp. Blot hybridisation showed that one set of ribosomal genes is located in each segment of the inverted repeat region and the sizes of these genes were accurately determined by measuring the dimensions of hybrids between the chloroplast rRNAs and the identified Sal and Eco fragments on electron micrographs: the genes for the 16S and 23S rRNAs contain 1530 bp and 2850 bp respectively and are separated by a spacer region of 2350 bp. The Bgl fragment of maize ctDNA known to contain the structural gene for the large-subunit (LS) of ribulose 1,5-bisphosphate carboxylase was used as a probe to locate the LS gene in wheat ctDNA. A small (2.8 kbp) Eco fragment was found to contain most of the wheat LS gene and is derived from the larger single-copy region, 23.5 kbp away from one segment of the inverted repeat and 54.8 kbp from the other.

B104, a new dispersed repeated gene family in Drosophila melanogaster and its analogies with retroviruses☆

Abstract The properties of B104, a new dispersed repeated gene family of Drosophila melanogaster are described. B104 was first discovered in a screen for genes differentially expressed at early embryonic development. The typical B104 element is 8.7 × 103 base-pairs in size and flanked by direct terminal repeats 429 base-pairs (bp) long. It is present in about 80 and 95 copies per haploid genome in Oregon R embryonic DNA and in Kcl cell line DNA, respectively, and accounts for about 0.4% of the D. melanogaster genome. While most of the B104 copies are closely conserved, a variant containing a 1.0 × 103 bp deletion as well as one containing a 2.4 × 103bp insertion have been found. B104 sequences are found in one large (about 8 × 103bp) and several short (0.8 to 1.2 × 103bp) transcripts. The DNA sequence of both terminal repeats in one element and of one terminal repeat in a second element was determined. In addition, two B104 complementary DNA clones originating from transcripts initiated and terminated, respectively, in the terminal repeat were also sequenced. The sequence data indicate: (1) that the B104 element is flanked by a 5 bp direct repeat; (2) that the sequence just internal to the left repeat shares homology with the transfer RNA primer binding site of retroviruses; and (3) that the B104 terminal repeat is redundantly transcribed in a manner typical of proviruses of vertebrate retroviruses.

APM‐1 , a novel human gene, identified by aberrant co‐transcription with papillomavirus oncogenes in a cervical carcinoma cell line, encodes a BTB/POZ‐zinc finger protein with growth inhibitory activity

Integration of human papillomavirus (HPV) DNA into the host cell genome is an important step in cervical carcinogenesis. In tumour cells with integrated HPV DNA, transcription of viral oncogenes E6 and E7 continues into the flanking cellular sequences thereby producing viral–cellular fusion transcripts. Analysis of cellular sequences flanking the integrated HPV68 DNA in the cervical carcinoma cell line ME180 revealed homozygosity of the mutant allele in ME180 cells. We speculated that this could indicate the existence of a cellular tumour suppressor gene in the integration region. We report here the identification of a novel human gene, named APM‐1, which is co‐transcribed with the HPV68 E6 and E7 genes and is present in the 3′‐cellular part of the ME180 viral–cellular fusion transcripts. The APM‐1 gene encodes a protein with a BTB/POZ domain and four zinc fingers, and is located at chromosome 18q21. APM‐1 transcripts are detected in normal cervical keratinocytes, but not in the majority of cervical carcinoma cell lines analysed. The APM‐1 gene caused a reduction of clonal cell growth in vitro of HeLa and CaSki tumour cells. These characteristics make APM‐1, the first novel human gene identified in a HPV integration region, a likely candidate for the postulated tumour suppressor gene.

The zinc finger gene Xblimp1 controls anterior endomesodermal cell fate in Spemann's organizer

The anterior endomesoderm of the early Xenopus gastrula is a part of Spemanns organizer and is important for head induction. Here we describe Xblimp1, which encodes a zinc finger transcriptional repressor expressed in the anterior endomesoderm. Xblimp1 represses trunk mesoderm and induces anterior endomesoderm in a cooperative manner with the pan‐endodermal gene Mix.1. Furthermore, Xblimp1 can cooperate with the BMP inhibitor chordin to induce ectopic heads, while a dominant‐negative Xblimp1 inhibits head formation. The head inducer cerberus is positively regulated by Xblimp1 and is able to rescue microcephalic embryos caused by dominant‐negative Xblimp1. Our results indicate that Xblimp1 is required for anterior endomesodermal cell fate and head induction.

Length measurements of RNA synthesized in vitro by Escherichia coli RNA polymerase.

Abstract A method for visualizing in vitro synthesized RNA in extended form still attached to the DNA template is described. Bacteriophage T4 gene 32 product is attached to the RNA and after fixation with glutaraldehyde the transcription complexes are prepared for electron microscopy by the Kleinschmidt technique. Secondary structure caused by partial complementarity of the RNA can be stretched out by the attachment of bacteriophage T4 gene 32 protein as is shown in the case of bacteriophage R17 RNA. The possibilities of the method are exemplified using T7 as a template for Escherichia coli RNA polymerase. Data for the position of the promoter and the extent of transcription in the region of early T7 messenger RNA are confirmed. In addition, we have demonstrated the presence of a weak promotor at the right-hand end of the DNA; as in the early region, the direction of RNA synthesis is from left to right. Using SV40 viral DNA as a template, up to six RNA chains can be synthesized in the presence of rifampicin. After synthesis for 15 minutes at 37 °C, the length of the RNA chains shows that the polymerase has transcribed at least four times around the DNA circle.