Xenopus laevis serum albumins are encoded in two closely related genes.

cDNA clones containing sequences complementary to Xenopus laevis albumin mRNA have been identified in a collection of cDNA clones made from poly(A)+ RNA prepared from male Xenopus laevis liver. Although all the albumin cDNA clones crosshybridise, restriction enzyme and heteroduplex analysis show that there are 2 closely related albumin mRNA sequences. The 2 albumin mRNAs are only mismatched by 8% but could be isolated by positive selection using stringent hybridization conditions. Oocytes injected with the 2 purified mRNAs, secreted either the 68,000 or 74,000 dalton albumin into the culture medium showing that the 2 albumins of X. laevis serum are encoded in the 2 closely related mRNAs. Measurements of the abundance of albumin mRNA show that the 2 albumin mRNAs together account for about 9% of total poly(A)+ RNA in male Xenopus laevis liver but the mRNA coding for the 74,000 dalton mRNA is about twice as abundant as that coding for the 68,000 dalton mRNA.     

The pattern of expression of the Xenopus laevis tadpole alpha-globin genes and the amino acid sequence of the three major tadpole alpha-globin polypeptides

We have isolated cDNA clones derived from three tadpole alpha-globin mRNAs of Xenopus laevis. The entire nucleotide sequence of the three mRNAs has been determined from the cDNA clones and is presented together with the deduced amino acid sequence of the encoded polypeptides. Two of the three polypeptide sequences are 96% homologous whilst the third sequence is highly diverged, with only a 72% homology. The three tadpole alpha-globin genes are all similarly diverged from the two X. laevis adult alpha-globin genes with which they display approximately 50% homology. Analysis of several independent clones from each class of tadpole alpha-globin sequence reveals a very high degree of coding region polymorphism for each of the three corresponding genes. Using the cloned DNA sequences as hybridisation probes, we have analysed the expression of the corresponding genes during larval development. We show that all three genes are activated simultaneously early in development and that thereafter all three are expressed at an approximately equivalent level. A fourth tadpole alpha-globin mRNA sequence, for which we do not have a cDNA clone, accumulates co-ordinately with the three major mRNA sequences but to a much lower concentration. This pattern of gene expression differs significantly from that of the tadpole beta-globin genes of X. laevis, despite the two classes of genes being closely linked in the genome.     

Characterization of cloned complementary DNA covering more than 6000 nucleotides (97%) of avian vitellogenin mRNA

The messenger RNA coding for chicken vitellogenin, a precursor of the egg-yolk proteins lipovitellin and phosvitin, is synthesized in the liver following estrogen injection. This mRNA is 6600 nucleotides long. We have previously reported the cloning and preliminary characterization of some cDNA fragments representing portions of the vitellogenin mRNA [Biochem. Biophys. Acta, 606, 34--46 (1980)]. In this paper we report the full characterization of a larger series of such clones, representing almost the entire length of the mRNA, by restriction endonuclease mapping, R-loop mapping, RNA-DNA hybridization and by translation in vitro of the RNA which hybridizes to the cloned DNA. From the results we conclude that the chicken vitellogenin mRNA, unlike that of Xenopus laevis, does not vary in sequence over most of its length, although some variations in the cDNA sequences were detected particularly in clones derived from the 3' terminus of the RNA. All sequence variants appear to be present in RNA prepared from single animals. The possible origins of these minor species are discussed. Furthermore, we describe a cDNA clone complementary to an mRNA which is about the same size as vitellogenin mRNA and which codes for an egg yolk protein antigenically related to lipovitellin. This mRNA is synthesized constitutively.     

Xenopus laevis integrins. Structural conservation and evolutionary divergence of integrin beta subunits

We report the sequences of cDNA clones for two different integrin beta subunits isolated from a Xenopus laevis neurula cDNA library. mRNAs corresponding to both genes are first detected at gastrulation. We show that these two beta subunits are very highly related (98% identity in amino acid sequence) and probably arose at the time of tetraploidization of the X. laevis genome around 50 million years ago. Comparison of these sequences with those of various other vertebrate integrin beta subunit establishes that all species analyzed to date contain a highly conserved integrin beta subunit (beta 1). The interspecies homologies within this class of integrin beta subunits (82-86% identity in amino acid sequence) are much greater than those among the three different beta subunits which are known in humans (40-48% identity in amino acid sequence). Analysis of the homologies clearly indicates duplication and divergence of this multigene family more than 500 million years ago prior to the appearance of the vertebrates. We also observe cross-hybridization between cDNA probes for chicken integrin beta subunits and genomic DNAs of several invertebrate species. Despite the divergence in sequence among different integrin beta subunits, certain features of their structure are remarkably conserved.     

Vitellogenin in Xenopus laevis is encoded in a small family of genes.

Vitellogenin, the yolk protein precursor, is produced in X. laevis liver from a 6.3 kilobase (kb) mRNA. Sequences of this mRNA have been transcribed into cDNA and cloned in E. coli. Some properties of 21 of these cloned DNAs, ranging in size from 1 to 3.7 kb, have been reported by Wahli et al. (1978b). This paper reports restriction endonuclease mapping, cross hybridization, heteroduplex mapping in the electron microscope and heteroduplex melting experiments with these DNAs. We conclude that the cloned DNAs fall into two main groups of sequences which differ from each other in approximately 20% of their nucleotides. Each main group contains two subgroups which differ from each other by about 5% sequence divergence. By hybridizing cloned DNAs with restricted genomic DNA, we showed that sequences corresponding to all four sequence groups are present in a single animal. Furthermore, we have obtained tentative evidence for the presence of large intervening sequences in genomic vitellogenin DNA. Analysis of R loop molecules demonstrated that all four sequences are present in the vitellogenin mRNA population purified from individual animals. While some alternate explanations are not entirely excluded, we suggest that vitellogenin is encoded by a small family of related genes in Xenopus.     

Vitellogenin genes and their products in closely and distantly related species of Xenopus

Plasma vitellogenins from two closely related species of Xenopus, X. laevis and X. borealis, and a more ancient species, X. tropicalis, exhibited the same size on gel electrophoresis and were immunologically related. Partial peptide maps of 125I-labelled plasma vitellogenins, however, revealed marked differences in th structure and organisation of vitellogenin in the three Xenopus species. Northern blot hybridisation of liver RNA from oestrogen-treated males and females, probed with cloned vitellogenin cDNA, revealed the presence of mRNA of the same size in the three species of Xenopus, which was absent in untreated male liver. Cell-free translation of total liver RNA showed the presence of functional mRNA coding for vitellogenin subunit of the same size (Mr congruent to 210,000). Restriction endonuclease digestion patterns of genomic DNA from the three Xenopus species, using cloned X. laevis vitellogenin cDNA as the hybridisation probe, revealed significant differences in the organisation of these genes, which occur at a higher multiplicity in X. laevis and X. borealis than in X. tropicalis. Thus, despite a high degree of conservation of size, overall sequence and immunological identity of vitellogenin genes and their products in the three species of Xenopus, there is a substantial structural rearrangement during evolution of Xenopus within this multigene family.     

Characterization of histone genes isolated from Xenopus laevis and Xenopus tropicalis genomic libraries

Using a cDNA clone for the histone H3 we have isolated, from two genomic libraries of Xenopus laevis and Xenopus tropicalis, clones containing four different histone gene clusters. The structural organization of X. laevis histone genes has been determined by restriction mapping, Southern blot hybridization and translation of the mRNAs which hybridize to the various restriction fragments. The arrangement of the histone genes in X. tropicalis has been determined by Southern analysis using X. laevis genomic fragments, containing individual genes, as probes. Histone genes are clustered in the genome of X. laevis and X. tropicalis and, compared to invertebrates, show a higher organization heterogeneity as demonstrated by structural analysis of the four genomic clones. In fact, the order of the genes within individual clusters is not conserved.     

Stage-specific keratins in Xenopus laevis embryos and tadpoles: the XK81 gene family

This report describes the isolation and characterization of genomic and cDNA clones which define a subfamily of type I keratins in Xenopus laevis whose expression is restricted to embryonic and larval stages. The XK81 subfamily, named after the prototype cDNA clone DG81, contains four members arranged in two pairs of closely homologous loci; they were named 81A1, A2, B1, and B2. Genomic clones were obtained representing all of these regions. The A1 gene has been completely sequenced together with approximately 1 kb of flanking sequences at each end; this gene corresponds to the previously reported cDNA clone 8128 (Jonas, E., T. D. Sargent, and I. B. Dawid, 1985, Proc. Natl. Acad. Sci. USA, 82:5413-5417). The B2 gene is represented by a partial cDNA clone, DG118. Upstream sequences and about half of the coding regions have been sequenced for the B1 and B2 genes, whereas the A2 locus has been identified on the basis of hybridization data and could be a gene or pseudogene. Genomic Southern blotting indicates that all members of the subfamily have been isolated. The keratin proteins encoded by the B1 and B2 genes are 96% homologous in the central rod domain, whereas A/B gene homology in this region is 81%. During development mRNAs derived from A and B genes accumulate coordinately during gastrula and neurula stages; in the tadpole, 81A mRNA decays rapidly, whereas 81B mRNA shows a second abundance peak, persists for most of tadpole life, and decays by metamorphosis. RNAs derived from the XK81 keratin subfamily are undetectable in the adult, where different type I keratin genes are expressed.     

Structure of Xenopus laevis ribosomal protein L32 and its expression during development.

cDNA clones for Xenopus laevis ribosomal protein L32 have been isolated and sequenced. The deduced amino acid sequence indicates that L32 is a basic protein of 110 amino acids, has a molecular weight of 12,603 and is homologous to the rat ribosomal protein L35. Using the cDNA clone as a probe to follow the expression of this gene during Xenopus development, it has been shown that the pattern of accumulation of this mRNA follows the one previously described for other ribosomal protein mRNAs during oogenesis and embryogenesis. The analysis of the utilization of L32 mRNA during embryogenesis shows that this is controlled by the translational regulation typical of other ribosomal protein mRNAs.     

Comparative nucleotide sequence analysis of two types of larval beta-globin mRNAs of Xenopus laevis.

The complete nucleotide sequence of the cDNA insert of the clone pXGL25 derived from the larval beta II-globin mRNA of Xenopus laevis has been determined. The sequence of 593 nucleotides represents part of the 5'nontranslated region, the coding region for 146 amino acids and the entire 3'nontranslated region. It diverges from the related larval beta I-sequence by 24.9% in the coding region. Alignment of the 5' and 3'nontranslated regions of the two related larval beta-sequences to maximum matching resulted in 31.2% and 46.7% divergence, respectively. Divergence between the corresponding adult and larval sequences considerably exceeds that of related larval sequences, suggesting that larval genes may have arisen by gene duplication prior to genome duplication. In contrast to mammalian beta-globin mRNAs, replacement and silent base substitutions are equally abundant, thus indicating less functional constraint on the larval Xenopus laevis beta-globin chains. The larval beta I- and beta II-globins diverge by 30.8% and show most variation in the alpha 1/beta 2-chain interaction sites.     

Two divergent cellular src genes are expressed in Xenopus laevis.

Genomic and cDNA clones of the X. laevis src gene have been isolated and characterized by hybridization and DNA sequence analyses. The haploid genome of X. laevis contains two src genes, which can be distinguished from one another by virtue of sequence divergence in the 3' untranslated regions. Both of the genes are functional as indicated by the fact that oocytes contain RNAs transcribed from each of the genes. The two genes each encode an RNA which is 3.3 kb in length, or twice the length required to encode the 60,000 dalton src protein (pp60). Sequence analysis of the cDNA clones revealed that nearly all of the non-coding sequence is located at the 3' end. The availability of sequence data from cDNA clones has also made it possible for the first time to identify with certainty the carboxyl terminal sequence of a cellular pp60 molecule.     

Isolation and characterization of sarcomeric actin genes expressed in Xenopus laevis embryos

A Xenopus laevis complementary DNA (cDNA) library prepared from messenger RNAs extracted from embryos has been screened for actin-coding sequences. Two cDNA clones corresponding to an alpha cardiac and an alpha skeletal muscle actin mRNA have been identified and characterized. From a genomic library, we have furthermore isolated the genes that correspond to the characterized cDNAs. In addition we have identified an actin processed gene which seems to be derived from a second type of skeletal muscle actin gene. Southern blot analysis of X. laevis DNA reveals that each of the three genes is present in at least two copies. In Xenopus tropicalis, a similar Southern blot analysis demonstrates that the three alpha actin genes exist as single copy. This result correlates with the genome duplication that has been proposed to have occurred recently in a X. laevis ancestor. A sequence comparison of the X. laevis cardiac and skeletal muscle actin cDNAs shows that the encoded peptides are highly conserved. Nevertheless, the numerous nucleotide changes at silent mutation sites suggest that the genes originated before the amphibia/reptile-bird divergence, more than 350 million years ago. Comparison of the promoters of the cardiac and skeletal actin genes, which are co-expressed in embryos, reveals a few common structural sequence elements.     

Histone gene number and organisation in Xenopus: Xenopus borealis has a homogeneous major cluster

Using a Xenopus laevis H4 cDNA clone as a probe we have determined that the numbers of H4 histone genes in Xenopus laevis and Xenopus borealis are approximately the same. These numbers are dependent on the hybridization stringency and we measure about 90 H4 genes per haploid genome after a 60 degrees C wash in 3 X SSC. Using histone probes from both Xenopus and sea urchin we have studied the genomic organization of histone genes in these two species. In all of the X.borealis individuals analyzed about 70% of the histone genes were present in a very homogeneous major cluster. These genes are present in the order H1, H2B, H2A, H4 and H3, and the minimum length of the repeated unit is 16kb. In contrast, the histone gene clusters in X.laevis showed considerable sequence variation. However two major cluster types with different gene orders seem to be present in most individuals. The differences in histone gene organization seen in species of Xenopus suggest that even in closely related vertebrates the major histone gene clusters are quite fluid structures in evolutionary terms.     

Molecular cloning of cDNA sequences coding for the major alpha- and beta-globin polypeptides of adult Xenopus laevis.

This report describes the synthesis and cloning of almost complete DNA copies of the mRNAs encoding the major alpha-globin and major beta-globin of X. laevis. Double-stranded globin cDNA was inserted into the PstI site of the plasmid pBR322 and two cloned recombinants (designated pXG6C1 and pXG8D2) were selected. These were shown to contain almost complete copies of X. laevis globin mRNA. Restriction enzyme maps were determined for each cDNA sequence using the established method of partial digestion of end labelled DNA. However, this procedure was modified such that isolation of individual DNA fragments was no longer required. Each plasmid was shown, by both hybrid arrested translation and filter selection of complementary RNA, to contain a sequence coding for one or other of the two major globin polypeptides. Sufficient DNA sequence information has been determined from each cDNA clone to demonstrate that pXG8D2 contains a beta-globin sequence and pXG6C1 contains an alpha-globin sequence.