Simple, directional cDNA cloning for in situ transcript hybridization screens.

Here, we describe a suppression PCR-based method for directional cloning of randomly primed cDNAs from small quantities of tissue. Synthesis of the first cDNA strand is conducted on oligonucleotide-coated magnetic beads. Synthesis of the second strand is accomplished using nonspecifically primed suppression PCR. This method is used to synthesize a cDNA library from zebrafish embryos at 6-9 h after fertilization. The sequencing of the clones and their use in an in situ hybridization screen to detect restricted patterns of gene transcription in zebrafish embryos showed that this method allows the rapid identification of genes that are important for development and genes that are expressed at levels undetectable by whole-mount in situ transcript hybridization. The random priming of cDNA alleviates the problems encountered in the identification of zebrafish genes from poly(dT)-primed cDNA clones caused by the long 3' UTRs frequently found in transcripts from this organism.

Evolutionary analysis of vertebrate Notch genes.

We have conducted an evolutionary analysis of Notch genes of the vertebrates Danio rerio and Mus musculus to examine the expansion and diversification of the Notch family during vertebrate evolution. The existence of multiple Notch genes in vertebrate genomes suggests that the increase in Notch signaling pathways may be necessary for the additional complexity observed in the vertebrate body plan. However, orthology relationships within the vertebrate Notch family indicate that biological functions are not fixed within orthologous groups. Phylogenetic reconstruction of the vertebrate Notch family suggests that the zebrafish notch1a and 1b genes resulted from a duplication occurring around the time of the teleost/mammalian divergence. There is also evidence that the mouse Notch4 gene is the result of a rapid divergence from a Notch3-like gene. Investigation of the ankyrin repeat region sequences showed there to be little evidence for gene conversion events between repeat units. However, relationships between repeats 2-5 suggest that these repeats are the result of a tandem duplication of a dual repeat unit. Selective pressure on maintenance of ankyrin repeat sequences indicated by relationships between the repeats suggests that specific repeats are responsible for particular biological activities, a finding consistent with mutational studies of the Caenorhabditis elegans gene glp-1. Sequence similarities between the ankyrin repeats and the region immediately C-terminal of the repeats further suggests that this region may be involved in the modulation of ankyrin repeat function.

Characterization and developmental expression of the amphioxus homolog of Notch (AmphiNotch): evolutionary conservation of multiple expression domains in amphioxus and vertebrates.

Notch encodes a transmembrane protein that functions in intercellular signaling. Although there is one Notch gene in Drosophila, vertebrates have three or more with overlapping patterns of embryonic expression. We cloned the entire 7575-bp coding region of an amphioxus Notch gene (AmphiNotch), encoding 2524 amino acids, and obtained the exon/intron organization from a genomic cosmid clone. Southern blot and PCR data indicate that AmphiNotch is the only Notch gene in amphioxus. AmphiNotch, like Drosophila Notch and vertebrate Notch1 and Notch2, has 36 EGF repeats, 3 Notch/lin-12 repeats, a transmembrane region, and 6 ankyrin repeats. Phylogenetic analysis places it at the base of all the vertebrate genes, suggesting it is similar to the ancestral gene from which the vertebrate Notch family genes evolved. AmphiNotch is expressed in all three embryonic germ layers in spatiotemporal patterns strikingly similar to those of all the vertebrate homologs combined. In the developing nerve cord, AmphiNotch is first expressed in the posteriormost part of the neural plate, then it becomes more broadly expressed and later is localized dorsally in the anteriormost part of the nerve cord corresponding to the diencephalon. In late embryos and larvae, AmphiNotch is also expressed in parts of the pharyngeal endoderm, in the anterior gut diverticulum, and, like AmphiPax2/5/8, in the rudiment of Hatschek's kidney. A comparison with Notch1 and Pax5 and Pax8 expression in the embryonic mouse kidney helps support homology of the amphioxus and vertebrate kidneys. AmphiNotch is also an early marker for presumptive mesoderm, transcripts first being detectable at the gastrula stage in a ring of mesendoderm just inside the blastopore and subsequently in the posterior mesoderm, notochord, and somites. As in sea urchins and vertebrates, these domains of AmphiNotch expression overlap with those of several Wnt genes and brachyury. These relationships suggest that amphioxus shares with other deuterostomes a common mechanism for patterning along the anterior/posterior axis involving a posterior signaling center in which the Notch and Wnt pathways and brachyury interact.

Nonspecific, nested suppression PCR method for isolation of unknown flanking DNA.

We report the development of a simple, sensitive and robust two-step PCR method for the isolation of unknown sequences flanking characterized regions of genomic DNA or cDNA. The method requires 100 bp or less of a known sequence upstream of an oligonucleotide primer binding site. A first round of suppression PCR is conducted at low stringency with a polymerase lacking exonuclease activity to generate a mixture of products including fragments of the desired flanking sequence that are often greater than 1 kb in length. The desired fragments are then amplified from the mixture in a second round of suppression PCR using an extended oligonucleotide in combination with a polymerase exhibiting exonuclease activity. These fragments are subsequently identified by hybridization with the 100 bp of known sequence or simply by cloning and sequencing. The method is widely applicable and allows isolation of novel cDNA from very low abundance transcripts.

A novel snoRNA (U73) is encoded within the introns of the human and mouse ribosomal protein S3a genes.

The mouse ribosomal protein S3a-encoding gene (mRPS3a) was cloned and sequenced in this study. mRPS3a shares identical exon/intron structure with its human counterpart. Both genes are split to six exons and exhibit remarkable conservation of the promoter region (68.8% identity in the 250 bp upstream of cap site) and coding region (the proteins differ in two amino acids). mRPS3a displays many features common to other r-protein genes, including the CpG-island at 5'-end of the gene, cap site within an oligopyrimidine tract and no consensus TATA or CAAT boxes. However, mRPS3a represents a rare subclass of r-protein genes that possess a long coding sequence in the first exon. Comparison of human and mouse S3a genes revealed sequence fragments with striking similarity within introns 3 and 4. Here we demonstrate that these sequences encode for a novel small nucleolar RNA (snoRNA) designated U73. U73 contains C, D and D' boxes and a 12-nucleotide antisense complementarity to the 28S ribosomal RNA. These features place U73 into the family of intron-encoded antisense snoRNAs that guide site-specific 2'-O-ribose methylation of pre-rRNA. We propose that U73 is involved in methylation of the G1739 residue of the human 28S rRNA. In addition, we present the mapping of human ribosomal protein S3a gene (hRPS3a) and internally nested U73 gene to the human chromosome 4q31.2-3.