Identification and characterization of a novel intelectin in the digestive tract of Xenopus laevis

The intelectin (Intl) family is a group of secretory lectins in chordates that serve multiple functions, including innate immunity, through Ca²⁺-dependent recognition of carbohydrate chains. Although six Intl family lectins have so far been reported in Xenopus laevis, none have been identified in the intestine. Using a monoclonal antibody to the Xenopus embryonic epidermal lectin (XEEL or Intl-1), I identified cross-reactive proteins in the intestines. The proteins were purified by affinity chromatography on a galactose-Sepharose column and found to be oligomers consisting of N-glycosylated 39 kDa and 40.5 kDa subunit peptides. N-terminal amino acid sequencing of these peptides, followed by cDNA cloning, identified two novel Intls (designated Intl-3 and Intl-4) that showed 59–79% amino acid identities with known Xenopus Intl family proteins. From the amino acid sequence, immunoreactivity, and properties of the recombinant protein, Intl-3 was considered the intestinal lectin identified by the anti-XEEL antibody. The purified Intl-3 protein could potentially bind to Escherichia coli and its lipopolysaccharides (LPS), and to Staphylococcus aureus and its peptidoglycans, depending on Ca²⁺. In addition, the Intl-3 protein agglutinated E. coli cells in the presence of Ca²⁺. Intraperitoneal injection of LPS increased the intestinal and rectal contents of Intl-3 and XCL-1 (or 35K serum lectin) proteins within three days; however, unlike XCL-1, Intl-3 was detectable in neither the sera nor the other tissues regardless of LPS stimulation. Immunohistochemical analyses revealed accumulation of the Intl-3 protein in mucus secretory granules of intestinal goblet cells. The results of this study suggest that Xenopus Intl-3 is involved in the innate immune protection of the digestive tract against bacterial infections.     

Isolation and characterization of a novel gene, xMADML, involved in Xenopus laevis eye development.

We have identified Xenopus MADM-like (xMADML), a Xenopus laevis gene related to the murine MADM and the human NRBP genes. xMADML is expressed throughout early development and is expressed most strongly in the developing lens and more weakly in the retina and other anterior tissues. We demonstrate that disruption of xMADML translation by means of morpholino injection results in impaired retina and lens development. Reciprocal transplantation of the presumptive lens ectoderm between morpholino-injected embryos and those injected solely with a dextran lineage tracer demonstrates that xMADML is necessary in both the lens and the retina for correct development of these eye tissues. Analysis of gene expression after knockdown of xMADML revealed significant alterations in the expression of some genes, including Pax6, xSix3, Sox2, and Sox3, suggesting that xMADML plays a role in regulating gene expression during development of the eye. This investigation is the first in vivo study examining the developmental role of this novel gene and reveals an important role of xMADML in eye tissue development and differentiation.     

大鼠睾丸精子发生中差异表达基因RSD5的克隆与表达分析

为了分离、克隆精子发生相关基因,深入了解精子发生的分子机理,本文以大鼠睾丸曲线精管显微分离结合DDRT－PCR的方法,所得的不同区段中差异表达的EST（expressed sequence tag)筛选大鼠睾丸cDNA文库,获得一个新的cDNA序列－RSD5。RSD5 cDNA全长1556bp,编码176个氨基酸,GenBank接收号为ASF146738。RSDS基因编码蛋白序列含有一个PEST基序,这是蛋白质快速降解的标志。Northern blot分析结果显示,RSD5在睾丸组织和脑组织表达量较高,且在不同发育天龄的睾丸组织中具有显著的表达差异性。RSD5基因的表达特征及其编码蛋白的PEST基序提示RSD5蛋白可能在精子发生中具有重要作用。     

Identification and characterization of centromeric sequences in Xenopus laevis

Centromeres play an essential function in cell division by specifying the site of kinetochore formation on each chromosome for mitotic spindle attachment. Centromeres are defined epigenetically by the histone H3 variant Centromere Protein A (Cenpa). Cenpa nucleosomes maintain the centromere by designating the site for new Cenpa assembly after dilution by replication. Vertebrate centromeres assemble on tandem arrays of repetitive sequences, but the function of repeat DNA in centromere formation has been challenging to dissect due to the difficulty in manipulating centromeres in cells. Xenopus laevis egg extracts assemble centromeres in vitro, providing a system for studying centromeric DNA functions. However, centromeric sequences in Xenopus laevis have not been extensively characterized. In this study, we combine Cenpa ChIP-seq with a k-mer based analysis approach to identify the Xenopus laevis centromere repeat sequences. By in situ hybridization, we show that Xenopus laevis centromeres contain diverse repeat sequences, and we map the centromere position on each Xenopus laevis chromosome using the distribution of centromere-enriched k-mers. Our identification of Xenopus laevis centromere sequences enables previously unapproachable centromere genomic studies. Our approach should be broadly applicable for the analysis of centromere and other repetitive sequences in any organism.

Accurate chromosome segregation during cell division requires the centromere, a nucleoprotein complex assembled on each chromosome that is essential for chromosome segregation. Centromeres provide the assembly site for the mitotic kinetochore that mediates microtubule attachment and error correction during mitosis (Foley and Kapoor 2013). Centromeres are defined epigenetically by the histone H3 variant, Centromere Protein A (Cenpa), the presence of which is both necessary and sufficient for centromere formation (Musacchio and Desai 2017). Unlike histone H3.1 nucleosomes, that are assembled as chromosomes replicate in S-phase, Cenpa nucleosomes are replenished after replication during the next G1 phase of the cell cycle. Cenpa nucleosomes in chromatin appear to epigenetically dictate the sites of new Cenpa incorporation, thereby providing a mechanism for self-maintenance (Zasadzińska and Foltz 2017).In humans, centromeres form on tandem repeats of an ∼171-bp DNA sequence termed ɑ-satellite. Each 171-bp monomer shares ∼60% sequence homology with other monomers. Tandem arrays of monomers are repeated in blocks of higher order repeats (HORs), resulting in long stretches of virtually identical repeat sequences (Willard and Waye 1987; Rudd et al. 2003; McNulty and Sullivan 2018). Investigation into the genetic features required to form stable human artificial chromosomes (HACs) identified repetitive ɑ-satellite DNA as sufficient for de novo centromere formation when introduced into human cells (Harrington et al. 1997; Ohzeki et al. 2015). These studies demonstrated that repetitive DNA promotes centromere formation in vertebrates.Perturbing centromere function in cells often leads to cell death; thus, cell-free systems using budding yeast and Xenopus laevis egg extracts have been invaluable for studying centromere and kinetochore assembly (Ng and Carbon 1987; Hyman et al. 1992; Sorger et al. 1994; Desai et al. 1997; Akiyoshi et al. 2010; Guse et al. 2011; Moree et al. 2011). Budding yeast centromeres are defined by a single 125-bp DNA sequence that is sufficient to recruit much of the centromere and kinetochore in cell extracts. As in humans, Xenopus laevis builds its centromeres on repetitive sequences (Edwards and Murray 2005), and thus Xenopus egg extract provides a unique system to study the functions of repetitive DNA in driving centromere formation. A 174-bp centromeric repeat has been previously identified in Xenopus laevis by chromatin immunoprecipitation of Cenpa followed by cloning and sequencing (Edwards and Murray 2005). This repeat sequence termed Frog centromere repeat 1 (Fcr1) forms large repetitive arrays and is AT rich, as are centromeric repeats from other vertebrates (Manuelidis 1978; McDermid et al. 1986; Melters et al. 2013; Sullivan et al. 2017). Fcr1 is detected on only 60%–70% of Xenopus laevis centromeres, suggesting that there must be other sequence elements that comprise Xenopus laevis centromeres. Xenopus laevis is an allotetraploid species: the genome is composed of two related subgenomes named the long (L) and short (S) based on the length of the homoeologous chromosomes (Session et al. 2016). Whether there is conservation of centromeric repeats within each subgenome or between homoeologous chromosomes remains unknown.In this study, we identified and characterized Cenpa-associated sequences in Xenopus laevis. We utilized a k-mer-based method that does not depend on an assembled reference genome and thus provides an unbiased approach to identify sequence motifs present at the centromere. Our results demonstrate the sequence diversity at active Xenopus laevis centromeres and enable future studies of the function of repetitive elements in centromere formation and function.     

Xenopus laevis pancreatic DNase I: purification and immunological characterization.

Deoxyribonuclease I (DNase I) was purified from Xenopus laevis pancreas to apparent electrophoretic homogeneity using a series of column chromatographies. The purified enzyme showed a molecular mass of about 36 kDa and maximum activity at pH 7.0-8.0, required divalent cations, Ca2+ and Mg2+, for its activity, and was inhibited by EDTA, EGTA and an antibody specific to the enzyme, but not by G-actin. The N-terminal amino acid sequence of the enzyme up to the 37th residue shared 38-44% homology with that of mammalian DNases I derived from bovine, ovine, porcine, rat, mouse, rabbit and human. A systematic survey of DNase I activity distribution in 20 different kinds of frog tissues showed that the pancreas and rectum produced higher amounts than other tissues. This is the first report concerning the purification and chemical and immunological characterization of frog pancreatic DNase I.     

Expression of rat ileal Na+-sulphate cotransport in Xenopus laevis oocytes: functional characterization

Small-intestinal sulphate absorption is a Na⁺-dependent process having its highest rate in the ileum; it involves brush-border membrane Na⁺-sulphate cotransport. Injection of rat ileal mRNA into Xenopus laevis oocytes induced Na⁺-dependent sulphate uptake in a dose-dependent manner, with no apparent effect on Na⁺-independent sulphate uptake. For mRNA-induced transport, the apparent K _m value for sulphate interaction was 0.6±0.2 mM and that for sodium interaction was 25±2 mM (Hill coefficient: 2.3±0.3). mRNA-induced transport, was inhibited by thiosulphate, but not by phosphate or 4,4,′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS). Using a rat renal Na⁺-sulphate cotransporter cDNA as a probe [NaSi-1; Markovich et al. (1993) Proc Natl Acad Sci USA 90:8073–8077], the highest hybridization signals (2.3 kb and 2.9 kb) were obtained in size fractions showing the highest expression of Na⁺-dependent sulphate transport in oocytes. Hybrid depletion experiments using antisense oligonucleotides (from the NaSi-1 cDNA sequence), provided further evidence that rat small-intestinal (ileal) Na⁺-sulphate cotransport is closely related to rat proximal-tubular brushborder membrane Na⁺-sulphate cotransport.     

Cloning and characterization of a novel feline IFN-omega.

The interferons (IFNs) are a large family of multifunctional secreted protein involved in antiviral defense, cell growth regulation, and immune activation. The human IFNs are used worldwide as antiviral drugs. Here, we present cDNAs encoding 13 novel feline IFN-omega (FeIFN-omega) subtypes that share 95%-99% amino acid sequence identity. FeIFN-omega2 and FeIFN-omega4 have seven additional amino acids at position 109 that are not present in other subtypes. Sequence identity of the present FeIFN proteins encoded by the 13 subtypes is approximately 57% compared with human IFN-omega (HuIFN-omega). All 13 FeIFN-omega subtypes were expressed in Escherichia coli using a periplasmic expression system. The antiviral activity of each product was evaluated in vitro. In addition, subtype FeIFN-omega2 was cytoplasm expressed in E. coli and secretion expressed in Pichia pastoris. The purified mature recombinant protein demonstrated significant antiviral activity on both homologous and heterologous animal cells in vitro.     

Cloning and characterization of voltage‐gated calcium channel alpha1 subunits in Xenopus laevis during development

Voltage‐gated calcium channels play a critical role in regulating the Ca²⁺ activity that mediates many aspects of neural development, including neural induction, neurotransmitter phenotype specification, and neurite outgrowth. Using Xenopus laevis embryos, we describe the spatial and temporal expression patterns during development of the 10 pore‐forming alpha1 subunits that define the channels' kinetic properties. In situ hybridization indicates that Ca_V1.2, Ca_V2.1, Ca_V2.2, and Ca_V3.2 are expressed during neurula stages throughout the neural tube. These, along with Ca_V1.3 and Ca_V2.3, beginning at early tail bud stages, and Ca_V3.1 at late tail bud stages, are detected in complex patterns within the brain and spinal cord through swimming tadpole stages. Additional expression of various alpha1 subunits was observed in the cranial ganglia, retina, olfactory epithelium, pineal gland, and heart. The unique expression patterns for the different alpha1 subunits suggests they are under precise spatial and temporal regulation and are serving specific functions during embryonic development. Developmental Dynamics 238:2891–2902, 2009. © 2009 Wiley‐Liss, Inc.     

Cloning and characterization of GABAA α subunits and GABAB subunits in Xenopus laevis during development

Gamma‐aminobutyric acid (GABA), the major inhibitory neurotransmitter in the adult nervous system, acts via two classes of receptors, the ionotropic GABA_A and metabotropic GABA_B receptors. During the development of the nervous system, GABA acts in a depolarizing, excitatory manner and plays an important role in various neural developmental processes including cell proliferation, migration, synapse formation, and activity‐dependent differentiation. Here we describe the spatial and temporal expression patterns of the GABA_A and GABA_B receptors during early development of Xenopus laevis. Using in situ hybridization and qRT‐PCR, GABA_A α2 was detected as a maternal mRNA. All other α‐subunits were first detected by tailbud through hatching stages. Expression of the various subunits was seen in the brain, spinal cord, cranial ganglia, olfactory epithelium, pineal, and pituitary gland. Each receptor subunit showed a distinctive, unique expression pattern, suggesting these receptors have specific functions and are regulated in a precise spatial and temporal manner. Developmental Dynamics 240:862–873, 2011. © 2011 Wiley‐Liss, Inc.     

Sodium dependence of the epithelial sodium conductance expressed in Xenopus laevis oocytes

The epithelial Na⁺ conductance was expressed in Xenopus laevis oocytes by injection of size-fractionated mRNA of bovine tracheal epithelium. Fractionation was achieved by sucrose density gradient centrifugation. Successful expression was analysed by recording current/voltage (I/V) curves in the presence and absence of amiloride (10 mol/l). The newly expressed conductance was half-maximally inhibited by 44 nmol/l amiloride and exhibited a selectivity for Na⁺ over K⁺ of 1401. I/V curves obtained at different extracellular Na⁺ concentrations ([Na⁺]_o) were subjected to a Goldman-fit analysis to obtain the relation between Na⁺ permeability (P _Na) and [Na⁺]_o. The data show that decreasing [Na⁺]_o from 85 mmol/l to 0.85 mmol/l increased P _Na by more than threefold, which is thought to reflect Na⁺ channel inhibition by increasing [Na⁺]_o. This effect clearly exceeded what can be attributed to concentration saturation of single Na⁺ channel conductance (Palmer and Frindt (1986) Proc Natl Acad Sci USA 83:2767). No correlation of inhibition with intracellular Na⁺ concentration was observed. Preservation of the [Na⁺]_o-dependent self-inhibition by the newly expressed Na⁺ conductance suggests that it is an intrinsic property of the Na⁺ channel protein, probably mediated by an extracellular Na⁺ binding site.     

Identification of genes expressed during Xenopus laevis limb regeneration by using subtractive hybridization.

Suppression polymerase chain reaction-based subtractive hybridization was used to identify genes that are expressed during Xenopus laevis hindlimb regeneration. Subtractions were done by using RNAs extracted from the regeneration-competent stage (stage 53) and regeneration-incompetent stage (stage 59) of limb development. Forward and reverse subtractions were done between stage 53 7-day blastema and stage 53 contralateral limb (competent stage), stage 59 7-day pseudoblastema and stage 59 contralateral limb (incompetent stage), and stage 53 7-day blastema and stage 59 7-day pseudoblastema. Several thousand clones were analyzed from the various subtracted libraries, either by random selection and sequencing (1,920) or by screening subtracted cDNA clones (6,150), arrayed on nylon membranes, with tissue-specific probes. Several hundred clones were identified from the array screens whose expression levels were at least twofold higher in experimental tissue vs. control tissue (e.g., blastema vs. limb) and selected for sequencing. In addition, primers were designed to assay several of the randomly selected clones and used to assess the level of expression of these genes during regeneration and normal limb development. Approximately half of the selected clones were differentially expressed, as expected, including several that demonstrate blastema-specific enhancement of expression. Three distinct categories of expression were identified in our screens: (1) clones that are expressed in both regeneration-competent blastemas and -incompetent pseudoblastemas, (2) clones that are expressed at highest levels in regeneration-competent blastemas, and (3) clones that are expressed at highest levels in regeneration-incompetent pseudoblastemas. Characterizing the role of each of these three categories of genes will be important in furthering our understanding of the process of tissue regeneration.     

Diversity of expressed V and J regions of immunoglobulin light chains in Xenopus laevis

In Xenopus laevis, two immunoglobulin light chain isotypes, designated L1 or ? and L2 or σ, have been identified. The genomic organization of the L1 locus has been described previously: a constant (C) gene segment is preceded by a joining (J) gene segment; in addition, there are many cross-hybridizing variable (V) gene segments. To evaluate the extent of sequence diversity of L1 V regions, we screened three cDNA libraries, constructed from mitogen-stimulated Xenopus splenocytes, with probes for the C or the J gene segment. Eighteen cDNA clones that contain complete or truncated V regions were chosen for sequence analysis. The C regions of all clones are identical or nearly identical to the genomic C gene segment; the V regions are greater than 80 % identical in nucleotide sequence and are presumably derived from a single family of V gene segments. Although framework regions are nearly identical, complementarity-determining regions are quite diverse. The expressed J segments fall into distinct groups, suggesting the presence of more than one germ-line J segment. Therefore, a genomic library was screened with a J region probe. A clone overlapping with the previously identified J- C clone, and containing four additional J gene segments, was isolated. All five J gene segments are very similar and three are identical in nucleotide sequence. Each of the three distinct germ-line J sequences is represented in the set of cDNA clones, suggesting that combinatorial diversification occurs; imprecision of V-J joining also appears to contribute to variability. Overall, these results suggest that the immunoglobulin repertoire in this species is not significantly restricted by a limitation in the diversity of light chain V regions.     

Cloning and characterization of Xenopus beta2-microglobulin

cDNAs for Xenopus beta2-microglobulin (beta2m), the obligatory light chain of most vertebrate Major Histocompatibility Complex (MHC) class I molecules, were isolated and ESTs were identified. Alignment of the deduced amino acid sequence to other species' beta2m showed that the overall structure is evolutionarily conserved, and phylogenetic analysis showed that the Xenopus beta2m sequence is intermediate between fish and bird/mammal beta2m. The Xenopus beta2m mRNA is expressed ubiquitously with highest expression in intestine, spleen, and thymus, correlating well with classical class Ia expression. beta2m mRNA and protein were also detected in Xenopus thymic tumor and kidney cell lines. Segregation analysis on a tetraploid Xenopus laevis family revealed two independently segregating, non-MHC-linked loci. As expected, only one locus was found in the diploid Xenopus tropicalis, strongly suggesting that the two beta2m loci in the tetraploid species were generated by genome-wide duplication, and did not undergo diploidization unlike many other MHC genes.