Evolutionary significance of an unusual chloroplast DNA inversion found in two basal angiosperm lineages

Two basal lineages of flowering plants possess an intergenic inversion in the chloroplast inverted repeat (IR), a region of the genome from which there have been few previous reports of this class of structural mutation. The size of the inversion (approximately 200 bp) places it in a class not previously seen in the plastid genome. The two lineages with the rearrangement, representatives of the orders Laurales and Nymphaeales, are not closely related and the inversion therefore probably arose independently in each group. The inversion is bordered by short, but highly conserved, inverted repeat motifs that were most likely associated with the inversion process. A stem-loop structure that involves these motifs may play a functional role in mRNA stability. It is seen in all optimal or nearly optimal predicted RNA foldings of the intergenic region. Received: 28 September / Accepted: 17 November 1999     

Dispersed repetitive sequences in the chloroplast genome of Douglas-fir

Summary Restriction mapping and DNA sequencing were used to characterize dispersed repetitive DNA in the chloroplast genome of Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco]. To map repeat families, chloroplast DNA (cpDNA) clones were hybridized at high stringency to one another and to cpDNA cut with restriction enzymes. Repeats are clustered in four regions of the genome and comprise at least six families. Sequence analysis of one repeat family shared among three XbaI fragments indicated the presence of a 633 by inverted repeat which contains a complete tRNA-Serine (GCU) gene and a highly conserved open reading frame (ORF 3.6). Both ends of this 633 bp dispersed repeat have a transposon-like combination of short direct and inverted repeats. One copy of the repeat flanks one of the endpoints of a major inversion which differentiates Douglas-fir from tobacco cpDNA. Dispersion of repetitive DNA by transposition, coupled with loss of the large inverted repeat, appears to have predisposed conifer cpDNA to a number of inversions. An 8 by (CATCTTTT) direct repeat in tobacco is located between two inverted sections in Douglas-fir; it may be a target sequence for homologous recombination.     

Characterization of a large inversion in the spinach chloroplast genome relative to Marchantia: a possible transposon-mediated origin

Summary A 7,022 by BamHI-EcoRI fragment, located in the inverted repeat of spinach chloroplast, has been sequenced. It contains a 2131 codon open reading frame (ORF) homologous to both tobacco ORFs 581 and 1708, and to Marchantia ORF 2136. Relative to the Marchantia chloroplast genome, spinach ORF 2131 is located at the end of a large inversion; the other end point is close to trnL, the position of which is the same in Marchantia, tobacco and spinach. In Marchantia, two 8 by direct repeats flanking two 10 by indirect repeats are present near the end points of the inversion. These repeats may result from a transposon-mediated insertion which would have facilitated the subsequent inversion. From a comparison of the gene organization of the spinach, tobacco, and Marchantia genomes in this region, we propose a step-wise process to explain the expansion of the inverted repeat from a Marchantia-like genome to the spinach/tobacco genome.     

The site of deletion of the inverted repeat in pea chloroplast DNA contains duplicated gene fragments

Summary Analysis of a nucleotide sequence from pea chloroplast DNA which spans the site of deletion of one copy of the large inverted repeat (IR) element suggests that the IR unit has been entirely deleted, but that the single-copy coding sequences which flanked it (trnH and ndh5) have been left intact. However, these flanking sequences have not simply been ligated together: between them there is instead a novel 200 by DNA sequence which includes two regions apparently derived by the duplication of fragments of the psbA and rbcL genes.     

Extensive sequence rearrangements in the chloroplast genomes of the green algaeChlamydomonas eugametos andChlamydomonas reinhardtii

Summary We have compared the overall sequence organization of chloroplast DNA (cpDNA) from the unicellular green algaeChlamydomonas eugametos andChlamydomonas reinhardtii. Cloned restriction fragments whose locations are known on the chloroplast genome of one or the other alga were hybridized to Southern blots of cpDNA digests from both algae and the positions of hybridization signals were used to align the two algal cpDNA restriction maps. In agreement with the important biological differences reported betweenC. eugametos andC. reinhardtii, we found extensive sequence rearrangements and low overall sequence homology between the two cpDNAs. To explain the striking contrast between our results and the remarkable conservation reported for the sequence and the organization of angiosperm cpDNAs containing inverted repeats, we suggest that the divergence between theC. eugametos andC. reinhardtii chloroplast genomes simply reflects a longer time of separate evolution relative to the angiosperm lineages. However, we cannot exclude the possibility that the algal cpDNAs rearranged faster than angiosperm cpDNAs.     

Organization and sequence of five tRNA genes and of an unidentified reading frame in the wheat chloroplast genome: evidence for gene rearrangements during the evolution of chloroplast genomes

Summary The genes for the initiator tRNA_CAU ^Met. tRNA_UCC ^Gly, tRNA_GGU ^Thr, tRNA_UUC ^Glu and tRNA_GUA ^Tyr and an open reading frame of 62 codons have been identified by sequencing a 2,358 by BamHl and a 1,378 by BamHI-Sst2 DNA fragments from wheat chloroplasts. A comparison of the organization of these five tRNA genes and of the open reading frame on the wheat, tobacco and spinach chloroplast genomes suggests that at least three genomic inversions must have occurred during the evolution of the wheat chloroplast genome from a spinach-like ancestor genome. Furthermore, it seems that in wheat the 91 by intergenic region between the genes for the initiator tRNA^Met and the gene for tRNA_UCC ^Gly is one end-point of the 20 kbp genomic inversion proposed by Palmer and Thompson in the case of maize (Palmer and Thompson 1982). A 119 bp duplication is located at this junction: the first copy comprises the 91 by of the intergenic region and the first 28 by of the tRNA^Met gene, the second copy is found downstream of the tRNA^Met gene.     

Molecular evolution of noncoding regions of the chloroplast genome in the Crassulaceae and related species

Universal primers were used for PCR amplification of three noncoding regions of chloroplast DNA (cpDNA) in order to study sequence-length variation in the Crassulaceae and in related species. Several length mutations were observed that are of diagnostic value for evolutionary relationships in the Crassulaceae and the Saxifragaceae. Length variation and sequence divergence in the intergenic spacer between the trnL (UAA) 3 exon and the trnF (GAA) gene among 15 species were studied in detail by nucleotide-sequence analysis. A total of 50 insertion/deletion mutations were observed, accounting for a spacer-length variation in the range of 228–360 bp. Eighteen short direct repeat motifs (4–11 bp) and two inverted repeat motifs (7–11 bp) were found to be associated with length variation. Phylogenetic analysis of the sequence data indicated a pattern of relationships that was largely consistent with a previous analysis of cpDNA restriction-site variation. Evaluation of the level of homoplasy in insertion/deletion mutations within a phylogenetic framework revealed that only 1 out of 34 length mutations longer than 2 bp must have had multiple origins. The feasibility of the noncoding chloroplast DNA regions for molecular evolutionary studies is discussed.     

A mutation hotspot in the chloroplast genome of a conifer (Douglas-fir: Pseudotsuga) is caused by variability in the number of direct repeats derived from a partially duplicated tRNA gene

We determined the DNA sequence of a 2.7-kb cpDNA XbaI fragment from douglas-fir [Pseudotsuga menziesii (Mirb.) Franco]. RFLPs revealed by the 2.7-kb XbaI clone were observed to vary up to 1 kb among species within the genus Pseudotsuga and up to 200 bp among trees of P. menziesii. The polymerase chain reaction (PCR) allowed the locus of polymorphism to be identified, and the variable region was then sequenced in a second Douglas-fir tree, a single tree of a related species, Japanese Douglas-fir (P. japonica), and in a species lacking a mutation hotspot in the region, Pinus radiata (Monterey pine). The locus of polymorphism is characterized by hundreds of base pairs of imperfect, tandem direct repeats flanked by a partially duplicated and an intact trnY-GUA gene. The duplication is direct in orientation and consists of 43 bp of the 3 end of trnY and 25 bp of its 3 flanking sequence. Tandem repeats show high sequence similarity to a 27-bp region of the trnY gene that overlaps one end of the duplication. The two trees of Douglas-fir sequenced differed by a single tandem repeat unit, whereas these trees differed from the Japanese Douglas-fir sequenced by approximately 34 repeat units. Repetitive DNA in the Pseudotsuga cpDNA hotspot was most likely generated at the time of the partial trnY gene duplication and these sequences expanded by slipped-strand mispairing and unequal crossingover.     

An analysis of the teratagenic effects that could possibly be due to alcohol consumption by pregnant mothers

It can be concluded that alcohol is definitely harmful to the developing fetus. The effect can manifest in various ways, the most extreme of which is a condition called Fetal Alcohol Syndrome (FAS). The diagnosis of maternal alcoholism leading onto cases of FAS is difficult due to absence of accurate diagnostic tests. The diagnosis of FAS in a child is easier by a proper examination. There is no specific treatment of FAS in a child. The only management is by institution of corrective and rehabilitative measures. The exact mechanism of the teratogenic action of alcohol is not known. It is probably due to the harmful effect of alcohol on the epiblast layer of the bilaminar germ disc. In the absence of adequate knowledge regarding FAS, not much can be done to remedy the deleterious effects of alcohol. Hence, a word of advice to all pregnant women is to avoid drinking during pregnancy.     

Cruciform-forming inverted repeats appear to have mediated many of the microinversions that distinguish the human and chimpanzee genomes

Submicroscopic inversions have contributed significantly to the genomic divergence between humans and chimpanzees over evolutionary time. Those microinversions which are flanked by segmental duplications (SDs) are presumed to have originated via non-allelic homologous recombination between SDs arranged in inverted orientation. However, the nature of the mechanisms underlying those inversions which are not flanked by SDs remains unclear. We have investigated 35 such inversions, ranging in size from 51-nt to 22056-nt, with the goal of characterizing the DNA sequences in the breakpoint-flanking regions. Using the macaque genome as an outgroup, we determined the lineage specificity of these inversions and noted that the majority (N = 31; 89%) were associated with deletions (of length beween 1-nt and 6754-nt) immediately adjacent to one or both inversion breakpoints. Overrepresentations of both direct and inverted repeats, ≥ 6-nt in length and capable of non-B DNA structure formation, were noted in the vicinity of breakpoint junctions suggesting that these repeats could have contributed to double strand breakage. Inverted repeats capable of cruciform structure formation were also found to be a common feature of the inversion breakpoint-flanking regions, consistent with these inversions having originated through the resolution of Holliday junction-like cruciforms. Sequences capable of non-B DNA structure formation have previously been implicated in promoting gross deletions and translocations causing human genetic disease. We conclude that non-B DNA forming sequences may also have promoted the occurrence of mutations in an evolutionary context, giving rise to at least some of the inversion/deletions which now serve to distinguish the human and chimpanzee genomes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Heteroplasmic suppression of an amber mutation in the Chlamydomonas chloroplast gene that encodes the large subunit of ribulosebisphosphate carboxylase/oxygenase

Summary The 18-5B and 18-7G mutants of Chlamydomonas reinhardtii lack ribulosebisphosphate carboxylase/oxygenase holoenzyme and contain nonsense mutations in the chloroplast gene that encodes the protein's large subunit. Spontaneous revertants of the 18-5B opal (UGA) mutant were found to be heteroplasmic in a previous study (Spreitzer et al. 1984). They appeared to contain both mutant and wild-type alleles of a suppressor gene within the chloroplast. However, revertants of the 18-7G amber (UAG) mutant could not be recovered spontaneously. In the present investigation, revertants of the opal and amber mutants were recovered after a mutagen treatment. Heteroplasmic suppression of the 18-7G amber mutant was observed, suggesting that heteroplasmic suppression may be a common genetic mechanism of polyploid genomes. Although a diversity of other revertant types was also observed, no significant alteration occurred in the oxygen sensitivity of ribulose-bisphosphate carboxylase acitivity.This research was supported in part by USDA Grant No. 85-CRCR-1-1563, and is published as Paper No. 8185, Journal Series, Nebraska Agricultural Research Division     

Prenatal diagnosis of fragile X syndrome by direct detection of the dynamic mutation due to an unstable DNA sequence

Yamauchi M, Nagata S, Seki N, Toyama Y, Harada N, Niikawa N, Masuno I, Kajii T, Hori T. Prenatal diagnosis of fragile X syndrome by direct detection of the dynamic mutation due to an unstable DNA sequence.
Clin Genet 1993: 44: 169–172. © Munksgaard, 1993
The fragile X syndrome is the most common familial form of mental retardation. The mutation causing the syndrome is dynamic mutation due to an unstable DNA (CCG)n repeat localized at Xq27.3. We have previously reported a PCR procedure to prepare a diagnostic probe, pPCRfx1, which can be used to determine the genotype of fragile X mutation individuals by Southern blot analysis. In the present study, pPCRfx1 was applied to the prenatal diagnosis, using chorionic villus cells, of a fetus which was at risk of having fragile X syndrome. In the PstI assay, the Southern blot showed the typical pattern of a female carrier with the full mutation. Analysis of the DNA methylation patterns by EcoRI + EagI assay showed that the EagI restriction site was not methylated on the mutated X chromosome of chorionic villi, but the sites were totally methylated in the brain and other tissues of the fetus. Thus the fetus was diagnosed to be a heterozygous female carrier of the dynamic mutation involved in the fragile X syndrome.     

Identification of a new DQB1 allele that appears to have been generated by an interallelic sequence exchange

The study reports the molecular characterization of a new DQB1 variant initially detected by unusual sequence-specific oligonucleotide (SSO) hybridization patterns in one Caucasoid individual. This new allele is identical to DQB1*0501 except for two silent nucleotide substitutions at codons 49 (GCA-->GCG) and 77 (AGG-->AGA). Compared with DQB1*0502 it differs in three nucleotides at codon 57 changing AGC (encoding Ser) to GTT (encoding Val). Considering the paternal genotype, it appears this new allele might have been generated by an interallelic sequence exchange between the two paternal DQB1 alleles.     

The earlier age of onset of malignancy in developing world is related to overall infection burden and could be due to the effect on telomere length

It is a common observation that many common cancers occur at a younger age in developing countries, like India. The cancer registry data provide incidence rate of different cancers, which suggest the same. Telomere shortening is involved in ageing of cells. Inflammation and infection result in telomere shortening in immune cells. The higher infection burden in developing countries might mean an earlier ageing of immune cells, resulting in decreased efficiency of immune surveillance and thus predisposing to cancer at an earlier age than seen in developed countries with lesser infection burden.     

Fusion of nearby inverted repeats by a replication-based mechanism leads to formation of dicentric and acentric chromosomes that cause genome instability in budding yeast

Large-scale changes (gross chromosomal rearrangements [GCRs]) are common in genomes, and are often associated with pathological disorders. We report here that a specific pair of nearby inverted repeats in budding yeast fuse to form a dicentric chromosome intermediate, which then rearranges to form a translocation and other GCRs. We next show that fusion of nearby inverted repeats is general; we found that many nearby inverted repeats that are present in the yeast genome also fuse, as does a pair of synthetically constructed inverted repeats. Fusion occurs between inverted repeats that are separated by several kilobases of DNA and share >20 base pairs of homology. Finally, we show that fusion of inverted repeats, surprisingly, does not require genes involved in double-strand break (DSB) repair or genes involved in other repeat recombination events. We therefore propose that fusion may occur by a DSB-independent, DNA replication-based mechanism (which we term “faulty template switching”). Fusion of nearby inverted repeats to form dicentrics may be a major cause of instability in yeast and in other organisms.     

Evidence that the anticonflict effect of midazolam in amygdala is mediated by the specific benzodiazepine receptors

The benzodiazepine midazolam produced an anticonflict effect in rats measured in a water lick paradigm following local injection into the basolateral and lateral complex of the amygdala. This effect of midazolam seems to involve specific benzodiazepine receptors, since the systemic injection of benzodiazepine antagonists Ro 15-1788, ZK 93426, FG 7142 and CGS 8216 produced strong antagonism of the effect of midazolam in doses not affecting the non-punished drinking behaviour.     

Translational reprogramming following UVB irradiation is mediated by DNA-PKcs and allows selective recruitment to the polysomes of mRNAs encoding DNA repair enzymes

UVB-induced lesions in mammalian cellular DNA can, through the process of mutagenesis, lead to carcinogenesis. However, eukaryotic cells have evolved complex mechanisms of genomic surveillance and DNA damage repair to counteract the effects of UVB radiation. We show that following UVB DNA damage, there is an overall inhibition of protein synthesis and translational reprogramming. This reprogramming allows selective synthesis of DDR proteins, such as ERCC1, ERCC5, DDB1, XPA, XPD, and OGG1 and relies on upstream ORFs in the 5′ untranslated region of these mRNAs. Experiments with DNA-PKcs-deficient cell lines and a specific DNA-PKcs inhibitor demonstrate that both the general repression of mRNA translation and the preferential translation of specific mRNAs depend on DNA-PKcs activity, and therefore our data establish a link between a key DNA damage signaling component and protein synthesis.     

Delta 9-tetrahydrocannabinol-induced catalepsy-like immobilization is mediated by decreased 5-HT neurotransmission in the nucleus accumbens due to the action of glutamate-containing neurons

Delta(9)-tetrahydrocannabinol (THC) has been reported to induce catalepsy-like immobilization, but the mechanism underlying this effect remains unclear. In the present study, in order to fully understand the neural circuits involved, we determined the brain sites involved in the immobilization effect in rats. THC dose-dependently induced catalepsy-like immobilization. THC-induced catalepsy-like immobilization is mechanistically different from that induced by haloperidol (HPD), because unlike HPD-induced catalepsy, animals with THC-induced catalepsy became normal again following sound and air-puff stimuli. THC-induced catalepsy was reversed by SR141716, a selective cannabinoid CB(1) receptor antagonist. Moreover, THC-induced catalepsy was abolished by lesions in the nucleus accumbens (NAc) and central amygdala (ACE) regions. On the other hand, HPD-induced catalepsy was suppressed by lesions in the caudate putamen (CP), substantia nigra (SN), globus pallidus (GP), ACE and lateral hypothalamus (LH) regions. Bilateral microinjection of THC into the NAc region induced catalepsy-like immobilization. This THC-induced catalepsy was inhibited by serotonergic drugs such as 5-hydroxy-L-tryptophan (5-HTP), a 5-HT precursor, and 5-methoxy-N,N-dimethyltryptamine (5-MeODMT), a 5-HT receptor agonist, as well as by anti-glutamatergic drugs such as MK-801 and amantadine, an N-methyl-d-aspartate (NMDA) receptor antagonist. THC significantly decreased 5-HT and glutamate release in the NAc, as shown by in vivo microdialysis. SR141716 reversed and MK-801 inhibited this decrease in 5-HT and glutamate release. These findings suggest that the THC-induced catalepsy is mechanistically different from HPD-induced catalepsy and that the catalepsy-like immobilization induced by THC is mediated by decreased 5-HT neurotransmission in the nucleus accumbens due to the action of glutamate-containing neurons.