Genevieve Pont-Kingdon

Mitochondrial Genomes of Anthozoa (Cnidaria)

The complete nucleotide sequences of the mitochondrial (mt) DNA molecules of the sea anemone, Metridium senile , and the sea pansy, Renilla kolikeri , have been analyzed. Both of these anthozoan mtDNAs contain genes for the same thirteen proteins and two rRNAs found in most other metazoan mtDNAs. However, only two and one tRNAs are encoded in M. senile and R. kolikeri mtDNAs, respectively. In M. senile mtDNA, two protein genes each contain a group I intron, and in R. kolikeri mtDNA, an extra gene for a protein with homology to a bacterial mismatch repair protein occurs. In these anthozoan mtDNAs: the only deviation from the standard genetic code is the use of TGA to specify tryptophan; all of the thirteen protein genes begin with ATG codons, and end with a complete codon; the one or two tRNAs that are encoded have a near-standard structure, and encoded s-rRNAs and l-rRNAs are closer in size and secondary structure potential to the homologous E. coli rRNAs than are any other metazoan mt-rRNAs. These observations suggest that almost all of the genetic novelties found in other metazoan mtDNAs arose after the cnidarian ancestral line diverged from the line that is common to all other present day Metazoa.

Direct molecular haplotyping by melting curve analysis of hybridization probes: beta 2-adrenergic receptor haplotypes as an example

Direct determination of the association of multiple genetic polymorphisms, or haplotyping, in individual samples is challenging because of chromosome diploidy. Here, we describe the ability of hybridization probes, commonly used as genotyping tools, to establish single nucleotide polymorphism (SNP) haplotypes in a single step. Three haplotypes found in the beta 2-adrenergic receptor (β2AR) gene and characterized by three different SNPs combinations are presented as examples. Each combination of SNPs has a unique stability, recorded by its melting temperature, even when intervening sequences from the template must loop out during probe hybridization. In the course of this study, two haplotypes in β2AR not described previously were discovered. This approach provides a tool for molecular haplotyping that should prove useful in clinical molecular genetics diagnostics and pharmacogenetic research where methods for direct haplotyping are needed.

Mitochondrial DNA of the sea anemone, Metridium senile (Cnidaria): Prokaryote-like genes for tRNAf-Met and small-subunit ribosomal RNA, and standard genetic code specificities for AGR and ATA codons

The nucleotide sequence of a segment of the mitochondrial DNA (mtDNA) molecule of the sea anemone Metridium senile (phylum Cnidaria, class Anthozoa, order Actiniaria) has been determined, within which have been identified the genes for respiratory chain NADH dehydrogenase subunit 2 (ND2), the small-subunit rRNA (s-rRNA), cytochrome c oxidase subunit II(COII), ND4, ND6, cytochrome b (Cyt b), tRNAf-Met, and the large-subunit rRNA (1-rRNA). The eight genes are arranged in the order given and are all transcribed from the same strand of the molecule. The overall order of the M. senile mt-genes differs from that of other metazoan mtDNAs. In M. senile mt-protein genes, AGA and AGG codons appear to have the standard genetic code specification of arginine, rather than serine as found for other invertebrate mt-genetic codes. Also, ATA has the standard genetic code specification of isoleucine. TGA occurs in three M. senile mt-protein genes and may specify tryptophan as in other metazoan, protozoan, and some fungal mt-genetic codes. The M. senile mt-rRNAf-Met gene has primary and secondary structure features closely resembling those of the Escherichia coli initiator tRNA, including standard dihydrouridine and TΨC loop sequences and a mismatch pair at the top of the aminoacyl stem. Determinations of the 5′ and 3′ end nucleotides of the M. senile mt-srRNAs indicated that these molecules have a homogenous size of 1,081 ntp, larger than any other known metazoan mt-s-rRNAs. Consistent with its larger size, the M. senile mt-s-rRNA can be folded into a secondary structure that more closely resembles that of the E. coli 16S rRNA than can any other metazoan mt-s-rRNA. These findings concerning M. senile mtDNA indicate that most of the unusual features regarding metazoan mt-genetic codes, rRNAs, and probably tRNAs developed after divergence of the Cnidarian line from the ancestral line common to other metazoa.

Target Specificity of the Endonuclease from the Xenopus laevis Non-Long Terminal Repeat Retrotransposon, Tx1L

ABSTRACT Elements of the Tx1L family are non-long terminal repeat retrotransposons (NLRs) that are dispersed in the genome ofXenopus laevis. Essentially all genomic copies of Tx1L are found inserted at a specific site within another family of transposable elements (Tx1D). This suggests that Tx1L is a site-specific retrotransposon. Like many (but not all) other NLRs, theXenopus element encodes an apparent endonuclease that is related in sequence to the apurinic-apyrimidinic endonucleases that participate in DNA repair. This enzyme is thought to introduce the single-strand break in target DNA that initiates transposition by the target-primed reverse transcription (TPRT) mechanism. To explore the issue of target specificity more fully, we expressed the polypeptide encoded by the endonuclease domain of open reading frame 2 from Tx1L (Tx1L EN) and characterized its cleavage capabilities. This endonuclease makes a specific nick in the bottom strand precisely at one end of the presumed Tx1L target duplication. Because this activity leaves a 5′-phosphate and 3′-hydroxyl at the nick, it has the location and chemistry required to initiate new insertion events by TPRT. Tx1L EN does not make a specific cut at a preferred target site for Tx1D elements, ruling out the alternative possibility that the composite Tx1L-Tx1D element moves as a unit under the control of functions encoded by Tx1L. Further characterization revealed that the endonuclease remains active for many hours at room temperature and that it is capable of enzymatic turnover. Scanning substitution mutagenesis located the recognition site for Tx1L EN within 10 bp surrounding the primary nick site. Implications of these features for natural transposition events are discussed.

The Alport Syndrome COL4A5 Variant Database

Alport Syndrome is a progressive renal disease with cochlear and ocular involvement. The most common form (∼80%) is inherited in an X‐linked pattern. X‐linked Alport Syndrome (XLAS) is caused by mutations in the type IV collagen alpha chain 5 (COL4A5). We have developed a curated disease‐specific database containing reported sequence variants in COL4A5. Currently the database archives a total of 520 sequence variants, verified for their position within the COL4A5 gene and named following standard nomenclature. Sequence variants are reported with accompanying information on protein effect, classification of mutation vs. polymorphism, mutation type based on the first description in the literature, and links to pertinent publications. In addition, features of this database include disease information, relevant links for Alport syndrome literature, reference sequence information, and ability to query by various criteria. On‐line submission for novel gene variants or updating information on existing database entries is also possible. This free online scientific resource was developed with the clinical laboratory in mind to serve as a reference and repository for COL4A5 variants.

Rapid Detection of Aneuploidy (Trisomy 21) by Allele Quantification Combined with Melting Curves Analysis of Single-Nucleotide Polymorphism Loci

BACKGROUND Molecular approaches for the detection of chromosomal abnormalities will allow the development of rapid, cost-effective screening strategies. We present here a molecular alternative for the detection of aneuploidies and, more specifically, trisomy 21. METHODS We used the quantitative value of melting curve analysis of heterozygous genetic loci to establish a relative allelic count. The two alleles of a given single-nucleotide polymorphism (SNP) were differentiated by thermodynamic stability with a fluorescently labeled hybridization probe and were quantified by relative areas of derivative melting curves detected after fluorescence resonance energy transfer. Heterozygous SNPs provided internal controls for the assay. RESULTS We selected six SNPs, heterozygous in at least 30% of a random population, to form a panel of informative loci in the majority of a random population. After normalization to a heterozygous control, samples segregated into three categories; nontrisomic samples had mean allele ratios of 0.96-1.09, whereas trisomic samples had mean ratios of 1.84-2.09 or 0.46-0.61, depending on which allele was duplicated. Within-run mean CVs of ratios were 6.5-27%, and between-assay mean CVs were 13-24%. CONCLUSIONS The use of melting curve analysis of multiple SNPs is an alternative to the use of small tandem repeats for the detection of trisomies. Because of the high density of SNPs, the approach may be specifically useful for very fine mapping of the regions of chromosome 21 that are critical for Down syndrome; it is also applicable to aneuploidies other than trisomy 21 and to specimens that are not amenable to cytogenetic analysis.

Design and Analytical Validation of Clinical DNA Sequencing Assays

CONTEXT DNA sequencing is the method of choice for mutation detection in many genes. OBJECTIVES To demonstrate the analytical accuracy and reliability of DNA sequencing assays developed in clinical laboratories. Only general guidelines exist for the validation of these tests. We provide examples of assay validation strategies for DNA sequencing tests. DESIGN We discuss important design and validation considerations. RESULTS The validation examples include an accuracy study to evaluate concordance between results obtained by the newly designed assay and analyzed by another method or laboratory. Precision (reproducibility) studies are performed to determine the robustness of the assay. To assess the quality of sequencing assays, several sequence quality measures are available. In addition, assessing the ability of primers to specifically and robustly amplify target regions before sequencing is important. CONCLUSION Protocols for validation of laboratory-developed sequencing assays may vary between laboratories. An example summary of a validation is provided.

Mitochondrial DNA of Hydra attenuata (Cnidaria): A Sequence That Includes an End of One Linear Molecule and the Genes for l-rRNA, tRNA f-Met , tRNA Trp , COII, and ATPase8

Abstract. The 3231-nucleotide-pair (ntp) sequence of one end of one of the two linear mitochondrial (mt) DNA molecules of Hydra attenuata (phylum Cnidaria, class Hydrozoa, order Anthomedusae) has been determined. This segment contains complete genes for tRNAf-Met, l-rRNA, tRNATrp, subunit 2 of cytochrome c oxidase (COII), subunit 8 of ATP synthetase (ATPase8), and the 5′ 136 ntp of ATPase6. These genes are arranged in the order given and are transcribed from the same strand of the molecule. As in two other cnidarians, the hexacorallian anthozoan Metridium senile and the octocorallian anthozoan Sarcophyton glaucum, the mt-genetic code of H. attenuata is near standard. The only modification appears to be that TGA specifies tryptophan rather than termination. Also as in M. senile and S. glaucum, the encoded H. attenuata mt-tRNAf-Met has primary and secondary structural features resembling those of Escherichia coli initiator tRNAt-Met. As the encoded mt-tRNATrp cannot be folded into a totally orthodox secondary structure, two alternative forms are suggested. The encoded H. attenuata mt-l-rRNA is 1738 nt, which is 451 nt shorter than the M. senile mt-l-rRNA. Comparisons of secondary structure models of these two mt-l-rRNAs indicate that most of the size difference results from loss of nucleotides in the H. attenuata molecule at a minimum of 46 locations, which includes elimination of six distinct helical elements.

A Novel Intron Element Operates Posttranscriptionally To Regulate Human N-myc Expression

ABSTRACT Precisely regulated expression of oncogenes and tumor suppressor genes is essential for normal development, and deregulated expression can lead to cancer. The human N-myc gene normally is expressed in only a subset of fetal epithelial tissues, and its expression is extinguished in all adult tissues except transiently in pre-B lymphocytes. The N-myc gene is overexpressed due to genomic amplification in the childhood tumor neuroblastoma. In previous work to investigate mechanisms of regulation of human N-mycgene expression, we observed that N-mycpromoter–chloramphemicol acelyltransferase reporter constructs containing sequences 5′ to exon 1 were active in all cell types examined, regardless of whether endogenous N-myc RNA was detected. In contrast, inclusion of the first exon and a portion of the first intron allowed expression only in those cell types with detectable endogenous N-myc transcripts. We investigated further the mechanisms by which this tissue-specific control of N-myc expression is achieved. Using nuclear run-on analyses, we determined that the N-myc gene is actively transcribed in all cell types examined, indicating a posttranscriptional mode of regulation. Using a series of N-myc intron 1 deletion constructs, we localized a 116-bp element (tissue-specific element [TSE]) within the first intron that directs tissue-specific N-myc expression. The TSE can function independently to regulate expression of a heterologous promoter-reporter minigene in a cell-specific pattern that mirrors the expression pattern of the endogenous N-myc gene. Surprisingly, the TSE can function in both sense and antisense orientations to regulate gene expression. Our data indicate that the human N-myc TSE functions through a posttranscriptional mechanism to regulate N-myc expression.

Sickle cell disease resulting from uniparental disomy in a child who inherited sickle cell trait

Sickle cell disease (SCD) is a classic example of a disorder with recessive Mendelian inheritance, in which each parent contributes one mutant allele to an affected offspring. However, there are exceptions to that rule. We describe here the first reported case of conversion of inherited sickle cell trait to SCD by uniparental disomy (UPD) resulting in mosaicism for SS and AS erythrocytes. A 14-year-old boy presented with splenomegaly and hemolysis. Although his father has sickle cell trait, his mother has no abnormal hemoglobin (Hb). DNA sequencing, performed to rule out Hb S/β-thalassemia, detected homozygous Hb SS. Further studies revealed mosaic UPD of the β-globin locus, more SS erythroid progenitors than AS, but a reverse ratio of erythrocytes resulting from the survival advantage of AS erythrocytes. This report exemplifies non-Mendelian genetics wherein a patient who inherited sickle cell trait has mild SCD resulting from postzygotic mitotic recombination leading to UPD.