Homoeologous chromosomes of Xenopus laevis are highly conserved after whole-genome duplication

It has been suggested that whole-genome duplication (WGD) occurred twice during the evolutionary process of vertebrates around 450 and 500 million years ago, which contributed to an increase in the genomic and phenotypic complexities of vertebrates. However, little is still known about the evolutionary process of homoeologous chromosomes after WGD because many duplicate genes have been lost. Therefore, Xenopus laevis (2n=36) and Xenopus (Silurana) tropicalis (2n=20) are good animal models for studying the process of genomic and chromosomal reorganization after WGD because X. laevis is an allotetraploid species that resulted from WGD after the interspecific hybridization of diploid species closely related to X. tropicalis. We constructed a comparative cytogenetic map of X. laevis using 60 complimentary DNA clones that covered the entire chromosomal regions of 10 pairs of X. tropicalis chromosomes. We consequently identified all nine homoeologous chromosome groups of X. laevis. Hybridization signals on two pairs of X. laevis homoeologous chromosomes were detected for 50 of 60 (83%) genes, and the genetic linkage is highly conserved between X. tropicalis and X. laevis chromosomes except for one fusion and one inversion and also between X. laevis homoeologous chromosomes except for two inversions. These results indicate that the loss of duplicated genes and inter- and/or intrachromosomal rearrangements occurred much less frequently in this lineage, suggesting that these events were not essential for diploidization of the allotetraploid genome in X. laevis after WGD.     

Evolution of duplicate genes in a tetraploid animal, Xenopus laevis

To understand the evolution of duplicate genes, we compared rates of nucleotide substitution between 17 pairs of nonallelic duplicated genes in the tetraploid frog Xenopus laevis with rates between the orthologous loci of human and rodent. For all duplicated X. laevis genes, the number of synonymous substitutions per site (dS) was greater than the number of nonsynonymous substitutions per site (dN), indicating that these genes are subject to purifying selection. There was also a significant positive correlation (r = 0.915) between dN for the X. laevis genes and dN for the mammalian genes, suggesting that, at the amino acid level, the X. laevis genes and the mammalian genes are under similar constraints. Results of relative-rate tests showed nearly equal rates of nonsynonymous substitution in each copy of the X. laevis genes; apparently there are similar constraints on both copies. No correlation was found between dS for the X. laevis genes and dS for the mammalian genes. There was a significant positive correlation both between members of pairs of duplicated X. laevis genes (r = 0.951) and between human and rodent orthologues (r = 0.854) with respect to third- position G+C content but no such relationship between the X. laevis genes and either of their mammalian orthologues. The results indicate that both copies of a duplicate gene can be subject to purifying selection and thus support the hypothesis of selection against all genotypes containing a null allele at either of two duplicate loci.      

The electrical block to polyspermy induced by an intracellular Ca2+ increase at fertilization of the clawed frogs,Xenopus laevis and Xenopus tropicalis

Polyspermy blocking, to ensure monospermic fertilization, is necessary for normal diploid development in most animals. We have demonstrated here that monospermy in the clawed frog, Xenopus tropicalis, as well as in X. laevis, is ensured by a fast, electrical block to polyspermy on the egg plasma membrane after the entry of the first sperm, which is mediated by the positive‐going fertilization potential. An intracellular Ca²⁺ concentration ([Ca²⁺]_i) at the sperm entry site was propagated as a Ca²⁺ wave over the whole egg cytoplasm. In the X. tropicalis eggs fertilized in 10% Steinberg's solution, the positive‐going fertilization potential of +27 mV was generated by opening of Ca²⁺‐activated Cl^?‐channels (CaCCs). The fertilization was completely inhibited when the egg's membrane potential was clamped at +10 mV and 0 mV in X. tropicalis and X. laevis, respectively. In X. tropicalis, a small number of eggs were fertilized at 0 mV. In the eggs whose membrane potential was clamped below ?10 mV, a large increase in inward current, the fertilization current, was recorded and allowed polyspermy to occur. A small initial step‐like current (IS current) was observed at the beginning of the increase in the fertilization current. As the IS current was elicited soon after a small increase in [Ca²⁺]_i, this is probably mediated by the opening of CaCCs. This study not only characterized the fast and electrical polyspermy in X. tropicalis, but also explained that the initial phase of [Ca²⁺]_i increase causes IS current during the early phase of egg activation of Xenopus fertilization.     

Chromosome complements of the genus Xenopus

The oytogenetic analysis of the genus Xenopus shows that X. laevis laevis, X. laevis petersi, X. laevis victorianus, X. (laevis) borealis, X. gilli, X. muelleri, and X. fraseri have chromosome numbers 2n=36; X. tropicalis has 20 (2n), X. (laevis) bunyoniensis 72 and X. ruwenzoriensis 108. This heterogeneity of the chromosome numbers is interesting as it represents new examples of polyploidy among Anurans. There are no big morphological differences among the karyotypes of the divers species, only the chromosomes with secondary constrictions vary considerably.     

Genetic evaluation of peroxisomal and cytosolic acetoacetyl-CoA thiolase isozymes in n-alkane-assimilating diploid yeast, Candida tropicalis

The n-alkane-assimilating diploid yeast, Candida tropicalis, possesses two acetoacetyl-CoA thiolase (Thiolase I) isozymes encoded by one allele: peroxisomal and cytosolic Thiolase Is encoded by both CT-T1A and CT-T1B. To clarify the function of peroxisomal and cytosolic. Thiolase Is, the site-directed mutation leading Thiolase I ΔC6 without a putative C-terminal peroxisomal targeting signal was introduced on CT-T1A locus in the ct-t1bΔ-null mutant. The C-terminus-truncated Thiolase I was active and solely present in the cytosol. Although the ct-t1aΔ/t1bΔ-null mutants showed mevalonate auxotrophy, the mutants having the C-terminus-truncated Thiolase I did not require mevalonate for growth, as did the strains having cytosolic Thiolase I. These results demonstrated that the presence of Thiolase I in the cytoplasm is indispensable for the sterol synthesis in this yeast. It is of greater interest that peroxisomal and cytosolic Thiolase I isozymes, products of the same genes, play different roles in the respective compartments, although further investigations will be necessary to analyze how to be sorted into peroxisomes and the cytosol.     

A Xenopus tropicalis oligonucleotide microarray works across species using RNA from Xenopus laevis

Microarrays have great potential for the study of developmental biology. As a model system Xenopus is well suited for making the most of this potential. However, Xenopus laevis has undergone a genome wide duplication meaning that most genes are represented by two paralogues. This causes a number of problems. Most importantly the presence of duplicated genes mean that a X. laevis microarray will have less or even half the coverage of a similar sized microarray from the closely related but diploid frog Xenopus tropicalis. However, to date, X. laevis is the most commonly used amphibian system for experimental embryology. Therefore, we have tested if a microarray based on sequences from X. tropicalis will work across species using RNA from X. laevis. We produced a pilot oligonucleotide microarray based on sequences from X. tropicalis. The microarray was used to identify genes whose expression levels changed during early X. tropicalis development. The same assay was then carried out using RNA from X. laevis. The cross species experiments gave similar results to those using X. tropicalis RNA. This was true at the whole microarray level and for individual genes, with most genes giving similar results using RNA from X. laevis and X. tropicalis. Furthermore, the overlap in genes identified between a X. laevis and a X. tropicalis set of experiments was only 12% less than the overlap between two sets of X. tropicalis experiments. Therefore researchers can work with X. laevis and still make use of the advantages offered by X. tropicalis microarrays.     

Developmental expression of the creatine kinase isozyme system of Xenopus: maternally derived CK-IV isoform persists far beyond the degradation of its maternal mRNA and into the zygotic expression period

The differential expression of the multilocus CK isozyme system throughout development of the two Xenopus species X. laevis and X. borealis was investigated. A cDNA containing the nearly complete coding sequence of the CK-IV subunit of X. laevis was isolated and sequenced. Early development of X. laevis proceeds with a stock of maternally derived CK-IV/IV isozyme. While the mRNA declines rapidly after fertilization and disappears before neurulation, maternal CK-IV/IV isozyme is active far beyond the onset of zygotic expression and is still detectable when tadpoles start feeding. Zygotic expression of CK-IV begins after neurulation, at stage 22/24, and seems to start simultaneously with that of another gene, CK-III. Modulation in the expression of these two genes and the appearance of two other isoforms, the CK-I and CK-II/III isozymes, take place during development in a tissue-specific manner. During metamorphosis, the CK phenotypes of eyes and skeletal musculature undergo additional changes. The final adult pattern only appears several weeks after metamorphosis. The presumed orthologous CK isozymes of X. borealis show a developmental profile similar to that of X. laevis, except that CK-II/II is equally present in oocytes and during early development, in addition to CK-IV/IV isozyme. These results show that the expression of each of the four CK genes of Xenopus is under differential developmental control.     

Resources and transgenesis techniques for functional genomics in Xenopus

Recent developments in genomic resources and high‐throughput transgenesis techniques have allowed Xenopus to ‘metamorphose’ from a classic model for embryology to a leading‐edge experimental system for functional genomics. This process has incorporated the fast‐breeding diploid frog, Xenopus tropicalis, as a new model‐system for vertebrate genomics and genetics. Sequencing of the X. tropicalis genome is nearly complete, and its comparison with mammalian sequences offers a reliable guide for the genome‐wide prediction of cis‐regulatory elements. Unique cDNA sets have been generated for both X. tropicalis and X. laevis, which have facilitated non‐redundant, systematic gene expression screening and comprehensive gene expression analysis. A variety of transgenesis techniques are available for both X. laevis and X. tropicalis, and the appropriate procedure may be chosen depending on the purpose for which it is required. Effective use of these resources and techniques will help to reveal the overall picture of the complex wiring of gene regulatory networks that control vertebrate development.     

Malate dehydrogenase isozymes in the longnose dace,Rhinichthys cataractae

The interspecies homology of dace supernatant (A₂, AB, B₂) and mitochondrial (C₂) malate dehydrogenase isozymes has been established through cell fractionation and tissue distribution studies. Isolated supernatant malate dehydrogenase (s-MDH) isozymes show significant differences in Michaelis constants for oxaloacetate and in pH optima. Shifts in s-MDH isozyme pH optima with temperature may result in immediate compensation for increase in ectotherm body pH with decrease in temperature, but duplicate s-MDH isozymes are probably maintained through selection for tissue specific regulation of metabolism.This research was supported in part by NSF Grant SM176-83974 and a grant from the Blakeslee Fund.     

Zebrafish transgenic constructs label specific neurons in Xenopus laevis spinal cord and identify frog V0v spinal neurons

A correctly functioning spinal cord is crucial for locomotion and communication between body and brain but there are fundamental gaps in our knowledge of how spinal neuronal circuitry is established and functions. To understand the genetic program that regulates specification and functions of this circuitry, we need to connect neuronal molecular phenotypes with physiological analyses. Studies using Xenopus laevis tadpoles have increased our understanding of spinal cord neuronal physiology and function, particularly in locomotor circuitry. However, the X. laevis tetraploid genome and long generation time make it difficult to investigate how neurons are specified. The opacity of X. laevis embryos also makes it hard to connect functional classes of neurons and the genes that they express. We demonstrate here that Tol2 transgenic constructs using zebrafish enhancers that drive expression in specific zebrafish spinal neurons label equivalent neurons in X. laevis and that the incorporation of a Gal4:UAS amplification cassette enables cells to be observed in live X. laevis tadpoles. This technique should enable the molecular phenotypes, morphologies and physiologies of distinct X. laevis spinal neurons to be examined together in vivo . We have used an islet1 enhancer to label Rohon‐Beard sensory neurons and evx enhancers to identify V0v neurons, for the first time, in X. laevis spinal cord. Our work demonstrates the homology of spinal cord circuitry in zebrafish and X. laevis , suggesting that future work could combine their relative strengths to elucidate a more complete picture of how vertebrate spinal cord neurons are specified, and function to generate behavior. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1007–1020, 2017     

Heterochromatin differentiation and phylogenetic relationship of the A genomes in diploid and polyploid wheats

Summary Heterochromatin differentiation, including band size, sites, and Giemsa staining intensity, was analyzed by the HKG (HCl-KOH-Giemsa) banding technique in the A genomes of 21 diploid (Triticum urartu, T. boeoticum and T. monococcum), 13 tetraploid (T. araraticum, T. timopheevi, T. dicoccoides and T. turgidum var. Dicoccon, Polonicum), and 7 cultivars of hexaploid (T. aestivum) wheats from different germplasm collections. Among wild and cultivated diploid taxa, heterochromatin was located mainly at centromeric regions, but the size and staining intensity were distinct and some accessions' genomes had interstitial and telomeric bands. Among wild and cultivated polyploid wheats, heterochromatin exhibited bifurcated differentiation. Heterochromatinization occurred in chromosomes 4A^t and 7A^t and in smaller amounts in 2A^t, 3A^t, 5A^t, and 6A^t within the genomes of the tetraploid Timopheevi group (T. araraticum, and T. timopheevi) and vice versa within those of the Emmer group (T. dicoccoides and T. turgidum). Similar divergence patterns occurred among chromosome 4A^a and 7A^a of cultivars of hexaploid wheat (T. aestivum). These dynamic processes could be related to geographic distribution and to natural and artifical selection. Comparison of the A genomes of diploid wheats with those of polyploid wheats shows that the A genomes in existing diploid wheats could not be the direct donors of those in polyploid wheats, but that the extant taxa of diploids and polyploids probably have a common origin and share a common A-genomelike ancestor.Contribution of the College of Agricultural Sciences, Texas Tech Univ. Journal No. T-4-233.     

Xenopus laevis RIC‐3 enhances the functional expression of the C. elegans homomeric nicotinic receptor,ACR‐16, in Xenopus oocytes

RIC‐3 enhances the functional expression of certain nicotinic acetylcholine receptors (nAChRs) in vertebrates and invertebrates and increases the availability of functional receptors in cultured cells and Xenopus laevis oocytes. Maximal activity of RIC‐3 may be cell‐type dependent, so neither mammalian nor invertebrate proteins is optimal in amphibian oocytes. We cloned the X. laevis ric‐3 cDNA and tested the frog protein in oocyte expression studies. X. laevis RIC‐3 shares 52% amino acid identity with human RIC‐3 and only 17% with that of Caenorhabditis elegans. We used the C. elegans nicotinic receptor, ACR‐16, to compare the ability of RIC‐3 from three species to enhance receptor expression. In the absence of RIC‐3, the proportion of oocytes expressing detectable nAChRs was greatly reduced. Varying the ratio of acr‐16 to X. laevis ric‐3 cRNAs injected into oocytes had little impact on the total cell current. When X. laevis, human or C. elegans ric‐3 cRNAs were co‐injected with acr‐16 cRNA (1 : 1 ratio), 100 μM acetylcholine induced larger currents in oocytes expressing X. laevis RIC‐3 compared with its orthologues. This provides further evidence for a species‐specific component of RIC‐3 activity, and suggests that X. laevis RIC‐3 is useful for enhancing the expression of invertebrate nAChRs in X. laevis oocytes.     

Simple embryo injection of long single‐stranded donor templates with the CRISPR/Cas9 system leads to homology‐directed repair in Xenopus tropicalis and Xenopus laevis

We report model experiments in which simple microinjection of fertilized eggs has been used to effectively perform homology‐directed repair (HDR)‐mediated gene editing in the two Xenopus species used most frequently for research: X. tropicalis and X. laevis. We have used long single‐stranded DNAs having phosphorothioate modifications as donor templates for HDR at targeted genomic sites using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR‐associated protein 9 (CRISPR/Cas9) system. First, X. tropicalis tyr mutant (i.e., albino) embryos were successfully rescued: partially pigmented tadpoles were seen in up to 35% of injected embryos, demonstrating the potential for efficient insertion of targeted point mutations. Second, in order to demonstrate the ability to tag genes with fluorescent proteins (FPs), we targeted the melanocyte‐specific gene slc45a2.L of X. laevis to label it with the Superfolder green FP (sfGFP), seeing mosaic expression of sfGFP in melanophores in up to 20% of injected tadpoles. Tadpoles generated by these two approaches were raised to sexual maturity, and shown to successfully transmit HDR constructs through the germline with precise targeting and seamless recombination. F1 embryos showed rescue of the tyr mutation (X. tropicalis) and tagging in the appropriate pigment cell‐specific manner of slc45a2.L with sfGFP (X. laevis).     

Isolation and some properties of DNA coding for tRNA1met from Xenopus laevis

DNA containing the reiterated genes for tRNA₁^met has been partially purified from Xenopus laevis by centrifugation in actinomycin C₁-CsCl and Ag⁺-Cs₂SO₄ gradients. These gradients separate the tRNA₁^met genes from those coding for tRNA₂^met and tRNA^val, thus confirming our earlier suggestion that these genes are not intermingled with each other (Clarkson, Birnstiel, and Purdom, 1973). The gradients also demonstrate the existence of a minor 5S DNA fraction which appears to differ from that previously isolated by Brown, Wensink, and Jordan (1971).When the enriched tDNA₁^met is digested to completion with either of the restriction endonucleases EcoRI or Hpa I, the tRNA₁^met genes are predominantly found within DNA fragments that are about 3100 base pairs long. A partial digestion with EcoRI shows that these fragments arise from the regular spacing of the enzyme restriction sites. The 3100 base pair EcoRI fragments are cleaved by Hpa I into fragments of two size classes, one of which is about 2200 base pairs long and contains the tRNA₁^met genes. The shorter fragments are about 700 base pairs long, and they appear to contain genes coding for at least one other kind of tRNA species. X. laevis tDNA₁^met thus comprises tandemly repeated DNA whose component parts show little if any length heterogeneity.     

Catalytic properties of alcohol dehydrogenase isozymes specified by duplicate genes in the diploid plant Stephanomeria exigua

The alcohol dehydrogenase (ADH) isozymes induced in flooded roots of the diploid plant Stephanomeria exigua are specified by tightly linked genes comprising a complex locus, Adh1. Individuals homozygous for a complex with two active genes which specify electrophoretically different subunits have three ADH-I isozymes, two intragenic homodimers and an intergenic heterodimer. Individual isozymes were partially purified from plants homozygous for several different Adh1 complexes and apparent K _m values for acetaldehyde, ethanol, NAD, and NADH and responses to temperature, pH, and two different alcohols were determined. The two homodimeric enzymes specified by a particular Adh1 complex generally differed in one or more of the properties studied, and in three of four cases, intergenic heterodimers differed significantly from intermediacy, often having lower K _m values than either homodimer. None of the isozymes studied could be considered greatly divergent or defective. Constraints on evolution of duplicate genes which form intergenic heterodimers are considered.     

Differential expression of creatine kinase isozymes during development of Xenopus laevis: An unusual heterodimeric isozyme appears at metamorphosis

The creatine kinase (CK) repertoire of Xenopus laevis, which is more complex than that of most other vertebrates, involves at least four genomic loci, all showing developmental and tissue-specific expression. The differential expression of this multilocus CK isozyme system was investigated by immunohistology. Specific monoclonal antibodies (mAb) against the three cytoplasmic CK isozymes of Xenopus laevis were isolated and characterized. Two of these mAbs, anti-CK-IV (DM16) and anti-CK-III (JRM4), were specific for CK-IV and CK-III subunits respectively, as well as for the corresponding homodimeric isozymes, CK-IV/IV and CK-III/III. Anti-CK-II (MRX7) mAb recognizes CK-II subunits and CK-II/III heterodimers; the homodimeric CK-II/III does not occur. Immunohistological localization on larval and adult tissue sections reveals that CK-IV epitopes, beside a generalized tissue distribution, are especially concentrated in the cytoplasm of some particular cells such as the photoreceptors in the outer segment of the retina, certain nerve cells of the spinal cord and spinal ganglia, and in larval hepatocytes. The CK-III III isozyme is specifically expressed in skeletal muscle, its appearance and accumulation occurring in parallel with myoblast differentiation. The CK-II antigen is detected first at the time of metamorphosis is skeletal muscles, as well as in the heart, eyes and brain. In striated musculature the expression of CK-II subunits during metamorphosis results in almost complete replacement of CK-III/III homodimers by CK-II/III heterodimers, as indicated by the progressive masking of CK-III epitope and the corresponding appearance of CK-II antigen. In the adult eyes, CK-II antigens localize at the same particular site of photoreceptors as do CK-IV antigens. Since that antigen represents a heterodimeric CK-II/III isozyme, this implies the activation of both CK-II and CK-III genes, none of which is expressed in larval retina.     

Functional expression of renal organic anion transport in Xenopus laevis oocytes

Secretion of organic anions by the kidney plays a critical role in the elimination of toxic agents from the body. Recent findings in isolated membranes and intact tissue have demonstrated the participation of multiple transport proteins in this process. As a first step toward molecular characterization of these proteins through expression cloning, the studies reported below demonstrate functional expression of both fumarate- and lithium-sensitive glutarate and probenecid-sensitive p-aminohippurate transport in Xenopus oocytes injected with rat kidney poly(A)⁺RNA. Maximal increase in substrate uptake over buffer-injected controls was reached by 5 days after mRNA injection. Expression of size-fractionated mRNA indicated that the active species with respect to both transport activities were in the range of 1.8 to 3.5 kb.