The homeotic protein HOXC13 is a member of human DNA replication complexes

The homeotic (and oncogenic) HOXC13 protein was shown to have an affinity for a DNA fragment corresponding to the sequence covered by the pre-replicative complex of the human lamin B2 replication origin. We show here that HOXC13 is a member of human replicative complexes. Our fluorescent fusion-protein data demonstrate that it co-localizes with replication foci of early-S cells and that this peculiar behaviour is driven by the homeodomain. By ChIP analysis we also show that HOXC13 binds the lamin B2 replication origin and the origins located near the TOP1 and MCM4 genes in asynchronously growing cells, whereas it does not bind these origins in G0 resting cells, consistently with its involvement in origin function.     

Homeotic proteins participate in the function of human-DNA replication origins

Recent evidence points to homeotic proteins as actors in the crosstalk between development and DNA replication. The present work demonstrates that HOXC13, previously identified as a new member of human DNA replicative complexes, is a stable component of early replicating chromatin in living cells: it displays a slow nuclear dynamics due to its anchoring to the DNA minor groove via the arginine-5 residue of the homeodomain. HOXC13 binds in vivo to the lamin B2 origin in a cell-cycle-dependent manner consistent with origin function; the interaction maps with nucleotide precision within the replicative complex. HOXC13 displays in vitro affinity for other replicative complex proteins; it interacts also in vivo with the same proteins in a cell-cycle-dependent fashion. Chromatin-structure modifying treatments, disturbing origin function, reduce also HOXC13–origin interaction. The described interactions are not restricted to a single origin nor to a single homeotic protein (also HOXC10 binds the lamin B2 origin in vivo). Thus, HOX complexes probably contribute in a general, structure-dependent manner, to origin identification and assembly of replicative complexes thereon, in presence of specific chromatin configurations.     

An Inducible DamID System for Profiling Interactions of Nuclear Lamina Protein Component Lamin B1 with Chromosomes in Mouse Cells

At the level of DNA organization into chromatin, there are mechanisms that define gene expression profiles in specialized cell types. Genes within chromatin regions that are located at the nuclear periphery are generally expressed at lower levels; however, the nature of this phenomenon remains unclear. These parts of chromatin interact with nuclear lamina proteins like Lamin B1 and, therefore, can be identified in a given cell type by chromatin profiling of these proteins. In this study, we created and tested a Dam Identification (DamID) system induced by Cre recombinase using Lamin B1 and mouse embryonic fibroblasts. This inducible system will help to generate genome-wide profiles of chromatin proteins in given cell types and tissues with no need to dissect tissues from organs or separate cells from tissues, which is achieved by using specific regulatory DNA elements and due to the high sensitivity of the method.     

Complete nucleotide sequence of pSMB 74, a plasmid encoding the production of pediocin AcH in Pediococcus acidilactici

Several Pediococcus acidilactici strains produce a plasmid-encoded bacteriocin, pediocin AcH. Previous studies have shown that this plasmid, designated as pSMB 74, encodes genes associated with the production of prepediocin, its post-translation processing to pediocin AcH, transmembrane translocation of these molecules, and immunity of producer cells against pediocin AcH. We report here the complete nucleotide sequence of pSMB 74. The plasmid has a total of 8877 bp. Four genes have been located on pSMB 74. The genes are arranged in a gene cluster of 3500 bp and share a common promoter and rho-independent stem-loop terminator. The four genes, each with independent ribosome binding sites (rbs), initiation and termination codons and spacer sequences in between, were designated as pap A, pap B, pap C and pap D and encode respectively for proteins of 62, 112, 174 and 724 amino acids. The results of this study can be useful either to introduce a suitable marker at a unique restriction site in pSMB 74 and use it as a vector or to clone the pap gene cluster in a suitable plasmid and transform desirable strains for pediocin AcH production. The gene sequence has been submitted to Gene Bank (Acc. No. U02482).     

Molecular cloning and expression of the functional gene encoding the M2 subunit of mouse ribonucleotide reductase: a new dominant marker gene.

Mammalian ribonucleotide reductase consists of two non-identical subunits, proteins M1 and M2. M2-related DNA sequences are present on mouse chromosomes 4, 7, 12 and 13. However, M2-overproducing mouse cells show amplification of a chromosome 12-specific, single 13 kb HindIII fragment, which probably represents the active gene. We have isolated this fragment from parental mouse cell DNA and used it to clone and characterize the functional M2 gene. The 5770 bp transcribed M2 sequence contains ten exons separated by nine 95-917 bp introns. The 501 bp of 5' flanking DNA is G + C rich and contains TTTAAA and CCAAT sequences as well as potential Sp1 binding sites. The M2-related sequence on chromosome 13, which contains only the last six exons and several internal rearrangements, is a pseudogene. Transfection of BALB/3T3 cells with the M2 gene resulted in stable transformants with a 10-fold reduction in sensitivity to hydroxyurea, compared to control cells. This confirmed that the cloned M2 genomic DNA represents the functional gene and conclusively establishes the link between hydroxyurea resistance and M2 expression in mammalian cells. M2 genomic DNA should be a valuable dominant, selectable marker for identifying and isolating stable co-transformants.     

TM7XN1, a novel human EGF-TM7-like cDNA, detected with mRNA differential display using human melanoma cell lines with different metastatic potential

We have identified a novel 3845 bp cDNA differentially expressed in a human melanoma metastasis model. Northern blot analysis showed expression in the poorly and intermediately metastasizing cell lines and a marked downregulation in the highly metastatic cell lines. Using RT-PCR expression was also seen in several other tumor cell lines and normal cell types of human origin. cDNA sequence analysis revealed an ORF of 687 amino acids containing seven putative transmembrane domains C-terminally and a long N-terminus. The gene was mapped to 16q13. Highest homology was observed with members of the EGF-TM7 subfamily of the secretin/calcitonin receptor family. We propose the delineation of a subfamily of TM7 proteins, LN-TM7, containing seven transmembrane proteins with a long N-terminal extracellular part.     

Aberrant splicing caused by the insertion of the B2 sequence into an intron of the complement C4 gene is the basis for low C4 production in H-2k mice.

The serum level of the fourth component of complement (C4) in mice bearing the H-2k haplotype is only 1/10 to 1/20 of that of non-H-2k mice. We have analyzed C4 cDNA clones from B10.BR(H-2k) mouse liver and found aberrant C4 cDNA which contained a 200-base pair (bp) insertion between the exon 13 and exon 14 encoded sequences in addition to the normal C4 cDNA. The 5' 148 bp and the 3' 52 bp of this insert were derived from the B2 sequence, the short interspersed repeats of mouse genome, and the central part of intron 13, respectively. Sequence analysis of intron 13 of the C4k gene showed the presence of a complete copy of a B2 consensus sequence. The structure of aberrant C4 mRNA indicated that the possible 3' splice site in the B2 sequence and the cryptic 5' splice site in intron 13 were used. Both the insertion of the B2 sequence into intron 13 and the presence of aberrant mRNA in the liver were specific to H-2k-bearing mice, suggesting that the aberrant splicing due to the B2 insertion is the basis for low C4 expression in H-2k mice.     

An 18-base-pair sequence in the mouse proalpha1(II) collagen gene is sufficient for expression in cartilage and binds nuclear proteins that are selectively expressed in chondrocytes.

The molecular mechanisms by which mesenchymal cells differentiate into chondrocytes are still poorly understood. We have used the gene for a chondrocyte marker, the proalpha1(II) collagen gene (Col2a1), as a model to delineate a minimal sequence needed for chondrocyte expression and identify chondrocyte-specific proteins binding to this sequence. We previously localized a cartilage-specific enhancer to 156 bp of the mouse Col2a1 intron 1. We show here that four copies of a 48-bp subsegment strongly increased promoter activity in transiently transfected rat chondrosarcoma (RCS) cells and mouse primary chondrocytes but not in 10T1/2 fibroblasts. They also directed cartilage specificity in transgenic mouse embryos. These 48 bp include two 11-bp inverted repeats with only one mismatch. Tandem copies of an 18-bp element containing the 3' repeat strongly enhanced promoter activity in RCS cells and chondrocytes but not in fibroblasts. Transgenic mice harboring 12 copies of this 18-mer expressed luciferase in ribs and vertebrae and in isolated chondrocytes but not in noncartilaginous tissues except skin and brain. In gel retardation assays, an RCS cell-specific protein and another closely related protein expressed only in RCS cells and primary chondrocytes bound to a 10-bp sequence within the 18-mer. Mutations in these 10 bp abolished activity of the multimerized 18-bp enhancer, and deletion of these 10 bp abolished enhancer activity of 465- and 231-bp intron 1 segments. This sequence contains a low-affinity binding site for POU domain proteins, and competition experiments with a high-affinity POU domain binding site strongly suggested that the chondrocyte proteins belong to this family. Together, our results indicate that an 18-bp sequence in Col2a1 intron 1 controls chondrocyte expression and suggest that RCS cells and chondrocytes contain specific POU domain proteins involved in enhancer activity.     

Biochemical characterization of Cdc6/Orc1 binding to the replication origin of the euryarchaeon Methanothermobacter thermoautotrophicus

Archaeal cell division cycle protein 6 (Cdc6)/Origin Replication Complex subunit 1 (Orc1) proteins share sequence homology with eukaryotic DNA replication initiation factors but are also structurally similar to the bacterial initiator DnaA. To better understand whether Cdc6/Orc1 functions in an eukaryotic or bacterial-like manner, we have characterized the interaction of two Cdc6/Orc1 paralogs (mthCdc6-1 and mthCdc6-2) with the replication origin from Methanothermobacter thermoautotrophicus. We show that while both proteins display a low affinity for a small dsDNA of random sequence, mthCdc6-1 binds tightly to a short duplex containing a single copy of a 13 bp sequence that is repeated throughout the origin. Surprisingly, sequence comparisons show that this 13 bp sequence is a minimized version of the Origin Recognition Box element found in many euryarchaeotal origins. Analysis of mthCdc6-1 mutants demonstrates that the helix–turn–helix motif in the winged-helix domain mediates the interaction with this sequence. Association of both mthCdc6/Orc1 paralogs with the duplex containing the minimized Origin Recognition Box fits to an independent binding sites model, but their interaction with longer DNA ligands is cooperative. Together, our data provide the first detailed biophysical characterization of the association of an archaeal DNA replication initiator with its origin. Our observations also indicate that the origin-binding properties of Cdc6/Orc1 proteins closely resemble those of bacterial DnaA.