Localization of single copy DNA sequences on G-banded human chromosomes by in situ hybridization

Recombinant lambda bacteriophage clone H3 containing a human DNA segment of 14.9 kb present in one or two copies per haploid genome was isolated. In situ hybridization to human metaphase chromosomes of the ³H-labeled cloned DNA resulted in highly significant labeling (53% of cells) of band p36 of chromosome 1, such that 22% of all chromosomal grains were located on this region. Hybridization was dependent upon the presence of dextran sulfate in the hybridization mixture and was not affected by repetitive DNA competitor. These results demonstrate localization of a single copy sequence on human metaphase chromosomes.     

Comparative analysis of Xenopus tropicalis and Xenopus laevis vitellogenin gene sequences

Analysis of cDNA clones synthesized from vitellogenin mRNA of X. tropicalis revealed three different types of cDNA clones, i.e. A, A* and B. A and A* clones have a sequence divergence of about 6% and are both related to X. laevis vitellogenin cDNAs of subgroup A1 as well as A2 with a sequence divergence of 6-9%. B clones however, are related to X. laevis cDNA clones of subgroup B1 and B2 with a sequence divergence of about 7%. While the A and B clones correspond to vitellogenin mRNAs of similar abundance, A* clone is complementary to a vitellogenin mRNA about 100 fold less abundant than A and B mRNAs although all three vitellogenin mRNAs are encoded by single copy genes. Furthermore, two forms of A* mRNA were found. One of the two is lacking an internal fragment of about 900 bp. Since this DNA fragment is highly repeated in the genome, we suggest that this A* clone was synthesized from a processing intermediate of the A* precursor vitellogenin mRNA.     

Direct visualization of single copy genes on banded metaphase chromosomes by nonisotopic in situ hybridization.

A rapid method is described for non isotopic in situ mapping of single copy genes directly on G-banded chromosomes by "one-step" regular light microscopy. It is based on hybridizing biotinylated probes to metaphase chromosomes. Biotin residues are detected by rabbit antibiotin antibody and anti-rabbit Ig labelled with peroxidase or colloidal gold. The peroxidase reaction product or colloidal gold signals are amplified by silver precipitation. The final product is a black silver dot at the gene locus on a purple G-banded chromosome. N-ras and alpha-1-antitrypsin genes have been mapped using plasmids with inserts of 1.5 and 1.3kb to 1p13.1 and the junction of 14q31/32 respectively. The signal to noise ratio in these experiments ranged from 32:1-46:1. This technology is at least as sensitive as radioisotopic in situ hybridization and gives results within 1 day of hybridization and has much better resolution. Additionally, genes are visualized by regular light microscopy without specialized techniques such as reflection contrast, fluorescence or phase microscopy. This methodology should facilitate more precise chromosomal gene localization.     

Fluorescence in situ hybridization of single copy transgenes in rice chromosomes

Summary Fluorescence in situ hybridization (FISH) is a powerful tool for visualizing the chromosomal location of targeted sequences and has been applied in many areas, including karyotyping, breeding and characterization of genes introduced into the plant genome. A simple, routine and sensitive FISH procedure was developed for localizing single copy genes in rice (Oryza sativa L.) metaphase chromosomes. We used digoxygenin-labeled endogenous or T-DNA sequences as small as 5.6 kb to probe corresponding endogenous sequences or the T-DNA insert in denatured rice metaphase chromosomes prepared from root meristem tissue. The hybridized probe sequence was labeled with cy3-conjugated anti-mouse IgG and visualized using fluorescence microscopy. Single copy and multiple copy introduced T-DNA sequences, as well as endogenous sequences, were localized on the chromosomes. The FISH protocol was effectively used to sereen the chromosomal location of introduced T-DNA and number of integration loci in rice.     

The c-SRC1 gene visualized by in situ hybridization on Xenopus laevis chromosomes

Fluorescent in situ hybridization followed by tyramide signal amplification was used to map the site of the c-SRC1 gene on XENOPUS LAEVIS chromosomes. Positive results were obtained with a cDNA probe of about 1 kb. The c-SRC1 gene is located in the subcentromeric region in the long arm of one of the acrocentric chromosomes of the G category (classified according to Graf and Kobel, 1991). The c-SRC1 gene and the XENOPUS major histocompatibility complex (MHC) 1b locus, which consists of 20 tandemly arranged gene copies, are situated on different chromosomes of the G category.     

Genome-wide analysis of gene expression during Xenopus tropicalis tadpole tail regeneration

Background

During liver development, intrahepatic bile ducts are thought to arise by a unique asymmetric mode of cholangiocyte tubulogenesis characterized by a series of remodeling stages. Moreover, in liver diseases, cells lining the Canals of Hering can proliferate and generate new hepatic tissue. The aim of this study was to develop protocols for three-dimensional visualization of protein expression, hepatic portal structures and human hepatic cholangiocyte tubulogenesis.

Results

Protocols were developed to digitally visualize portal vessel branching and protein expression of hepatic cell lineage and extracellular matrix deposition markers in three dimensions. Samples from human prenatal livers ranging from 7 weeks + 2 days to 15½ weeks post conception as well as adult normal and acetaminophen intoxicated liver were used. The markers included cytokeratins (CK) 7 and 19, the epithelial cell adhesion molecule (EpCAM), hepatocyte paraffin 1 (HepPar1), sex determining region Y (SRY)-box 9 (SOX9), laminin, nestin, and aquaporin 1 (AQP1). Digital three-dimensional reconstructions using CK19 as a single marker protein disclosed a fine network of CK19 positive cells in the biliary tree in normal liver and in the extensive ductular reactions originating from intrahepatic bile ducts and branching into the parenchyma of the acetaminophen intoxicated liver. In the developing human liver, three-dimensional reconstructions using multiple marker proteins confirmed that the human intrahepatic biliary tree forms through several developmental stages involving an initial transition of primitive hepatocytes into cholangiocytes shaping the ductal plate followed by a process of maturation and remodeling where the intrahepatic biliary tree develops through an asymmetrical form of cholangiocyte tubulogenesis.

Conclusions

The developed protocols provide a novel and sophisticated three-dimensional visualization of vessels and protein expression in human liver during development and disease.     

Evolution of ribosomal RNA gene copy number on the sex chromosomes of Drosophila melanogaster

A diverse array of cellular and evolutionary forces--including unequal crossing-over, magnification, compensation, and natural selection--is at play modulating the number of copies of ribosomal RNA (rRNA) genes on the X and Y chromosomes of Drosophila. Accurate estimates of naturally occurring distributions of copy numbers on both the X and Y chromosomes are needed in order to explore the evolutionary end result of these forces. Estimates of relative copy numbers of the ribosomal DNA repeat, as well as of the type I and type II inserts, were obtained for a series of 96 X chromosomes and 144 Y chromosomes by using densitometric measurements of slot blots of genomic DNA from adult D. melanogaster bearing appropriate deficiencies that reveal chromosome-specific copy numbers. Estimates of copy number were put on an absolute scale with slot blots having serial dilutions both of the repeat and of genomic DNA from nonpolytene larval brain and imaginal discs. The distributions of rRNA copy number are decidedly skewed, with a long tail toward higher copy numbers. These distributions were fitted by a population genetic model that posits three different types of exchange events--sister-chromatid exchange, intrachromatid exchange, and interchromosomal crossing-over. In addition, the model incorporates natural selection, because experimental evidence shows that there is a minimum number of functional elements necessary for survival. Adequate fits of the model were found, indicating that either natural selection also eliminates chromosomes with high copy number or that the rate of intrachromatid exchange exceeds the rate of interchromosomal exchange.     

A genetic map of Xenopus tropicalis

Posttranslational modifications constitute a major field of emerging biological significance as mounting evidence demonstrates their key role in multiple physiological processes. Following in the footsteps of protein phosphorylation studies, new modifications are being shown to regulate protein properties and functions in vivo. Among such modifications, an important role belongs to protein arginylation — posttranslational tRNA-mediated addition of arginine, to proteins by arginyltransferase, ATE1. Recent studies show that arginylation is essential for embryogenesis in many organisms and that it regulates such important processes as heart development, angiogenesis, and tissue morphogenesis in mammals. This review summarizes the key data in the protein arginylation field since its original discovery to date.     

Localization of repetitive DNA sequences on in vitro Xenopus laevis chromosomes by primed in situ labeling (PRINS)

Xenopus laevis is an important reference model organism used in many vertebrate studies. Gene mapping in X. laevis, in comparison to other reference organisms, is in its early stages. Few studies have been conducted to localize DNA sequences on X. laevis chromosomes. Primed in situ labeling (PRINS) is a recently developed innovative tool that has been used to locate specific DNA sequences in various organisms. PRINS has been reported to have increased sensitivity compared to other in situ hybridization techniques. In the present study, PRINS was first used to label the location of telomeres at the ends of in vitro X. laevis chromosomes. The terminal location was as expected from in vivo reports, however, the overall amount seemed to decrease in the in vitro chromosomes. Once the PRINS technique was optimized, this technique was used to determine the chromosomal location of the satellite 1 repetitive sequence, which is an important sequence in X. laevis development. The sequence was observed on the interstitial regions of the majority of the chromosomes similar to the in vivo locations reported. In contrast to the telomeric sequence, the amount of sequence appeared to increase in the satellite 1 sequence. PRINS was found to be useful in the localization of repetitive DNA sequences in the X. laevis genome.     

Localization of the ornithine aminotransferase gene and related sequences on two human chromosomes

Summary We have used a full length cDNA clone to determine the chromosomal location ofthegene encoding human ornithine aminotransferase (OAT), a mitochondrial matrix enzyme. Southern blot analysis of ScaI-digested DNA from 34 human-mouse somatic cell hybrids revealed 11 human fragments. Three fragments mapped to chromosome 10q23-10qter, confirming the previous provisional assignment of the functional gene to this autosome by analysis of OAT expression in somatic cell hybrids (O'Donnell et al. 1985). The remaining eight fragments were assigned to the X chromosome, and regionally assigned to Xp21-Xp11 by use of an X-chromosome mapping panel. These X chromosome sequences could represent pseudogenes, or related members of a multigene family. Two of the X chromosome fragments are alternate alleles of a restriction fragment length polymorphism (RFLP) making this OAT-related locus an excellent genetic marker. The RFLP may now be used to determine any possible relationship between this locus and several X-linked eye defects.     

Construction of BAC library for the amphibian Xenopus tropicalis

Xenopus tropicalis has become an alternative model to the amphibian Xenopus laevis because it is better suited for genetic and genomic studies. We have constructed a genomic BAC library consisting of over 100,000 clones from sperm of Xenopus tropicalis. Analysis by pulsed field gel electrophoresis of representative BAC clones indicated the average size of insert DNA to be 100 kb, and we estimated the library covers 6 times the Xenopus tropicalis genome of 1.7 x 10(9) base pairs. To evaluate the BAC library, we attempted to isolate BAC clones which contain a protocadherin gamma (Pcdh gamma) gene and found that the isolated BAC clones are assembled as two separate contigs. This result suggests the presence of at least two clusters for the Pcdh gamma gene in the genome of X. tropicalis.     

Localization by in situ hybridization of a low copy chimaeric resistance gene introduced into plants by direct gene transfer

Summary An in situ hybridization method was developed for detecting single or low copy number genes in metaphase chromosomes of plants. Using as a probe ³H-labelled plasmid pABDI, which confers kanamycin resistance (Km^r) to transformed cells. DNA introduced into the plant genome by direct gene transfer was detected with a high efficiency: about 60% to 80% of interphase and metaphase plates showed a strong signal. The insertion site of the Km^r gene in two independent transformants was localised on different homologous chromosome pairs. This result independently confirmed previous genetic data which had indicated that transformed DNA was integrated into plant chromosomes in single blocks.     

Comparison of induction during development between Xenopus tropicalis and Xenopus laevis

Several in vitro systems exist for the induction of animal caps using growth factors such as activin. In this paper, we compared the competence of activin-treated animal cap cells dissected from the late blastulae of Xenopus tropicalis and Xenopus laevis. The resultant tissue explants from both species differentiated into mesodermal and endodermal tissues in a dose-dependent manner. In addition, RT-PCR analysis revealed that organizer and mesoderm markers were expressed in a similar temporal and dose-dependent manner in tissues from both organisms. These results indicate that animal cap cells from Xenopus tropicalis have the same competence in response to activin as those from Xenopus laevis.     

Xenopus tropicalis nodal-related gene 3 regulates BMP signaling: an essential role for the pro-region

In vertebrates, nodal-related genes are crucial for specifying mesendodermal cell fates. Six nodal-related genes have been identified in Xenopus, but only one, nodal, has been identified in the mouse. The Xenopus nodal-related gene 3 (Xnr3), however, lacks the mesoderm-inducing activity of the other five nodal-related genes in Xenopus, and can directly induce neural tissue in animal caps by antagonizing BMP signals. In this study, we isolated three clones of the Xenopus (Silurana) tropicalis nodal-related gene 3 (Xtnr3) and analyzed their function. The Xtnr3 genes show high homology to Xnr3 and have the same activity. Southern blot and genomic PCR analyses indicate that the X. tropicalis genome has duplications in the Xtnr3 gene sequences and our three clones represent separate gene loci. We also found a partial clone of Xtnr3 that coded for the N-terminal part of its pro-region. Surprisingly, this sequence also induced neural tissue by antagonizing BMP signals, and its coded protein physically associated with BMP4 mature protein. Furthermore, we showed that the pro-region of Xnr5 has the same activity. Together, these findings indicate that the pro-region of nodal-related genes acts antagonistically towards BMP signals, which identifies a novel mechanism for the inhibition of BMP signaling.     

Defining synphenotype groups in Xenopus tropicalis by use of antisense morpholino oligonucleotides

To identify novel genes involved in early development, and as proof-of-principle of a large-scale reverse genetics approach in a vertebrate embryo, we have carried out an antisense morpholino oligonucleotide (MO) screen in Xenopus tropicalis, in the course of which we have targeted 202 genes expressed during gastrula stages. MOs were designed to complement sequence between −80 and +25 bases of the initiating AUG codons of the target mRNAs, and the specificities of many were tested by (i) designing different non-overlapping MOs directed against the same mRNA, (ii) injecting MOs differing in five bases, and (iii) performing “rescue” experiments. About 65% of the MOs caused X. tropicalis embryos to develop abnormally (59% of those targeted against novel genes), and we have divided the genes into “synphenotype groups,” members of which cause similar loss-of-function phenotypes and that may function in the same developmental pathways. Analysis of the expression patterns of the 202 genes indicates that members of a synphenotype group are not necessarily members of the same synexpression group. This screen provides new insights into early vertebrate development and paves the way for a more comprehensive MO-based analysis of gene function in X. tropicalis.