A Xenopus laevis creatine kinase isozyme (CK-III/III) expressed preferentially in larval striated muscle: cDNA sequence, developmental expression and subcellular immunolocalization.

A cDNA containing the nearly complete coding sequence of CK-III subunit of X. laevis was isolated, sequenced and further identified by comparing the tissue distribution of CK-III/III isozyme with that of its messenger. Comparison of CK-III deduced amino acid sequence with other CK sequences published reveals its close homology to M-CK subunits. Results using both cDNA probes and monoclonal antibodies specific for CK-III subunits indicate that the appearance and the accumulation of CK-III occur in parallel with myoblast differentiation. Moreover, subcellular immuno-histolocalization shows that CK-III/III isozyme is especially concentrated on larval myofibres at the level of A-bands.

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.

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.

Purification and characterization of cytoplasmic creatine kinase isozymes of Xenopus laevis.

The soluble creatine kinase isozymes CK-II, CK-III, and CK-IV from Xenopus laevis have been purified to apparent homogeneity and their subunits characterized by means of molecular weight, peptide pattern, and dissociation-reassociation experiments. CK-III and CK-IV are homodimeric isozymes whose subunits are distinct in both molecular weight (42,000 and 41,000, respectively) and Staphylococcus aureus V8 peptide pattern. In dissociation-reassociation experiments, those two subunits do form active heterodimeric isozymes with one another or with rabbit M-CK subunits. Hybrid CK-III/IV isozymes occur also during embryonic differentiation and in adult heart muscle, whereas most other adult tissues contain only homodimeric CK-III or CK-IV isozymes. The CK-II isozyme is a heterodimer composed of one CK-III subunit and another subunit specific to CK-II (Mr = 41,000). Neither in vivo nor in vitro does this subunit seem able to form homodimers or heterodimers with CK-IV and rabbit M-CK subunits. If we take into account the apparent association of CK-I isozyme with cellular organelles, these results corroborate earlier statements and suggest that the CK isozyme system of X. laevis is encoded by at least four differentially regulated genomic loci.

The Z-chromosome is involved in the regulation of H-W (H-Y) antigen gene expression in Xenopus.

H-W (H-Y) antigen was investigated in diploid, triploid, and tetraploid Xenopus hybrids. These hybrids differ from each other with respect to their sex chromosome constitution-they all have a single W chromosome but one to three Z chromosomes. The H-W antigen concentration is reduced with increasing numbers of Z chromosomes. In the diploid and triploid hybrids the single W chromosome is able to guarantee ovary development, but among the tetraploids both male and female animals occur. However, both sexes have identical H-W antigen titers in the somatic tissues. It can be concluded that in these tetraploid hybrids with a ZZZW sex chromosome complement, the amount of H-W antigen is too low to sustain ovary development in all cases. Our results clearly indicate that in Xenopus a Z-coded gene is involved in the regulation of H-W antigen gene expression. In man and mouse there is evidence that such a regulatory gene is located on the X chromosome. Because of the concordance of man, mouse, and Xenopus we hypothesize that not only was the H-Y (H-W) antigen gene conserved during evolution but also the genes engaged in its regulation.

Two transitions of haemoglobin expression in Xenopus: from embryonic to larval and from larval to adult.

Electrophoretic analyses of haemoglobin and globin phenotypes in families of Xenopus borealis and Xenopus l. laevis revealed two developmental haemoglobin transitions during ontogeny. The first transition occurs at the developmental stage when tadpoles begin to feed. It is characterized by the decline of embryonic-specific globins in favour of novel, tadpole-specific globins (X. borealis) correlated to changes in the haemoglobin pattern. We suppose that this switch results from the replacement of a primitive, ventral blood island-dependent erythrocyte population by tadpole erythrocytes from other erythropoietic sites. Several other globin chains and haemoglobins are present in both young tadpoles and throughout larval life. The second, well-known transition occurs during metamorphosis, where all tadpole haemoglobins are replaced by adult haemoglobins composed of entirely different globin chains.

Lactate dehydrogenase of Xenopus laevis laevis and Xenopus borealis depends on a multiple gene system.

Lactate dehydrogenase isozymes have been reinvestigated in Xenopus laevis laevis and Xenopus borealis. High resolution zymograms of various organs demonstrate that in both species the LDH isozymes are governed basically by a three-gene system: Ldh-a coding for positively charged polypeptide, and Ldh-b and Ldh-c for negatively charged polypeptides (at pH 8.9). These three LDH subunits when assembled in tetramers show differential sensitivity to heat inactivation; the C4 homotetramer is the most labile isozyme. Xenopus is thus similar to Osteichthyes, birds, and mammals that also have a three-gene system for LDH. With respect to tissue specific isozyme expression, Xenopus resembles the more primitive families of bony fish. Superimposed on this three-gene system is a probable gene duplication for both LDH-a and Ldh-b. Heterotetrameric isozymes are formed between the various subunits leading to multibanded zymograms with a total of at least 21 distinct zones of LDH activity. The expression of genes is tissue specific not only for the basic genes, but also for their presumed duplicates. Since both species of Xenopus are ancient tetraploids, duplication of Ldh genes is not surprising.

Developmental capacity of aneuploid Xenopus species hybrids.

Diploid as well as triploid Xenopus interspecific hybrids generate aneuploid eggs because of the presence, at meiosis, of univalent chromosomes which are presumably distributed at random. Zygotes obtained from such eggs, fertilized by either normal or UV-irradiated sperm, were analysed for their developmental capacities. All monosomics die in the course of embryogenesis, whereby optimum capacities correspond closely with those observed in monosomic mammalian embryos, especially in mice. In contrast, hyperdiploid Xenopus are relatively viable: although many die exhibiting the 'haploid syndrome' or various other abnormalities, 8% of them reach metamorphosis, and 1-2% become adults. Of the latter, the karyotype was established in 13 individuals. Among them, 8-16 supernumerary chromosomes were found to be present.

Two new polyploid Xenopus species from western Uganda.

2 new species of the anuran genus Xenopus have been found in western Uganda: X. ruwenzoriensis sp.n. with the hexaploid chromosome number of 108 in the Semliki Valley, west of the Ruwenzori, and X. species nova with the tetraploid chromosome number of 72 in and near lake Bunyoni.