Minisatellites, microsatellites, and short random oligonucleotides all uncover highly polymorphic DNA fingerprint patterns in Southern analysis of genomic DNA that has been digested with a restriction enzyme having a 4-bp specificity. The polymorphic nature of the fragments is attributed to tandem repeat number variation of embedded minisatellite sequences. This explains why DNA fingerprint fragments are uncovered by minisatellite probes, but does not explain how it is that they are also uncovered by microsatellite and random oligonucleotide probes. To clarify this phenomenon, we sequenced a large bovine genomic BamHI restriction fragment hybridizing to the Jeffreys 33.6 minisatellite probe and consisting of small and large Sau3A-resistant subfragments. The large Sau3A subfragment was found to have a complex architecture, consisting of two different minisatellites, flanked and separated by stretches of unique DNA. The three unique sequences were characterized by sequence simplicity, that is, a higher than chance occurrence of tandem or dispersed repetition of simple sequence motifs. This complex repetitive structure explains the absence of Sau3A restriction sites in the large Sau3A subfragment, yet provides this subfragment with the ability to hybridize to a variety of probe sequences. It is proposed that a large class of interspersed tracts sharing this complex yet simplified sequence structure is found in the genome. Each such tract would have a broad ability to hybridize to a variety of probes, yet would exhibit a dearth of restriction sites. For each restriction enzyme having 4-bp specificity, a subclass of such tracts, completely lacking the corresponding restriction sites, will be present. On digestion with the given restriction enzyme, each such tract would form a large fragment.(ABSTRACT TRUNCATED AT 250 WORDS)
CdxA is a homeobox gene of the caudal type that was previously shown to be expressed in the endoderm-derived gut epithelium during early embryogenesis. Expression of the CDXA protein was studied during intestine morphogenesis from stage 11 (13 somites) to adulthood in the chicken. The CDXA protein can be detected during all stages of gut closure, from stage 11 to 5 days of incubation, and is mainly localized to the intestinal portals, the region where the splanchnopleure is undergoing closure. In this region, which represents the transition between the open and closed gut, the CDXA protein is restricted to the endoderm-derived epithelium. At about day 5 of incubation, the process of formation of the previllous ridges begins, which marks the beginning of the morphogenesis of the villi. From this stage to day 11 expression of CDXA is localized to the epithelial lining of the intestine. In parallel, a gradual increase in CDXA protein expression begins in the mesenchyme that is close in proximity to the CDXA-positive endoderm. Maximal CDXA levels in the mesenchyme are observed at day 9 of incubation. During days 10 and 11 CDXA levels in the mesenchyme remain constant, and by day 12 CDXA becomes undetectable in these cells and the epithelium again becomes the main site of expression. From day 12 of incubation until adulthood the CDXA protein is present in the intestinal epithelium. Until day 18 of incubation expression can be detected along the whole length of the villus with a stronger signal at the tip. With hatching the distribution along the villi changes so that the main site of CDXA protein expression is at the base of the villi and in the crypts. The transient expression of CDXA in the mesenchyme between days 5 and 11 may be related to the interactions taking place between the mesenchyme and the epithelium that ultimately result in the axial specification of the alimentary canal and the differentiation of its various epithelia. The main CDXA spatial distribution during morphogenesis suggests a tight linkage to the formation and differentiation of the intestinal epithelium itself. CDXA appears to play a role in the morphogenetic events leading to closure of the alimentary canal. During previllous ridge formation the CDXA protein is transiently expressed in the mesenchymal cells thought to provide instructive interactions for the regionalization and differentiation of the gut epithelium.(ABSTRACT TRUNCATED AT 400 WORDS)
Sax1 (previously CHox3) is a chicken homeobox gene belonging to the same homeobox gene family as the Drosophila NK1 and the honeybee HHO genes. Sax1 transcripts are present from stage 2 H&H until at least 5 days of embryonic development. However, specific localization of Sax1 transcripts could not be detected by in situ hybridization prior to stage 8-, when Sax1 transcripts are specifically localized in the neural plate, posterior to the hindbrain. From stages 8- to 15 H&H, Sax1 continues to be expressed only in the spinal part of the neural plate. The anterior border of Sax1 expression was found to be always in the transverse plane separating the youngest somite from the yet unsegmented mesodermal plate and to regress with similar dynamics to that of the segregation of the somites from the mesodermal plate. The posterior border of Sax1 expression coincides with the posterior end of the neural plate. In order to study a possible regulation of Sax1 expression by its neighboring tissues, several embryonic manipulation experiments were performed. These manipulations included: removal of somites, mesodermal plate or notochord and transplantation of a young ectopic notochord in the vicinity of the neural plate or transplantation of neural plate sections into the extraembryonic area. The results of these experiments revealed that the induction of the neural plate by the mesoderm has already occurred in full primitive streak embryos, after which Sax1 is autonomously regulated within the spinal part of the neural plate.
The chicken homebox containing gene, CdxA (formerly CHox-cad), was previously shown to be expressed during gastrulation. Localization of CdxA transcripts by in situ hybridization to tissue sections revealed that, during gastrulation, expression of this gene exhibits a posterior localization along the primitive streak. The transcripts are localized to epiblast cells in the vicinity of the primitive streak, to cells of the primitive streak itself and in the definitive endoderm as it replaces the hypoblast. In order to study in greater detail the pattern of expression of the CdxA gene during gastrulation, we expressed the full-length CdxA protein as a fusion protein in E. coli and generated monoclonal antibodies against it. Chicken embryos at different stages of gastrulation were processed for whole-mount immunohistochemical localization of the protein using anti-CdxA antibodies. Once the pattern of expression in the whole embryo was determined, the same embryos were sectioned to determine the identity of the cells expressing the CdxA protein. Detailed analysis of the CdxA protein in embryos, from the onset of primitive streak formation to the beginning of the tail bud stage (stages 2 to 10), has shown different patterns of expression during primitive streak elongation and regression. The CdxA protein is initially detected at the posterior marginal zone and the expression moves rostrally into the primitive streak during mid-streak stages. As the primitive streak elongates, the CdxA stripe of expression moves anteriorly. By definitive streak stages, the CdxA stripe of expression delineates a position along the anterior-posterior axis in the primitive streak. CdxA, like its Drosophila homologue cad, is expressed during gastrulation in a stripe localized to the posterior region of the embryo. These observations suggest that CdxA as a homebox gene may be part of a regulatory network coupled to axial determination during gastrulation in the early chick embryo.
The DNA binding specificity of the chicken homeodomain protein CDXA was studied. Using a CDXA-glutathione-S-transferase fusion protein, DNA fragments containing the binding site for this protein were isolated. The sources of DNA were oligonucleotides with random sequence and chicken genomic DNA. The DNA fragments isolated were sequenced and tested in DNA binding assays. Sequencing revealed that most DNA fragments are AT rich which is a common feature of homeodomain binding sites. By electrophoretic mobility shift assays it was shown that the different target sequences isolated bind to the CDXA protein with different affinities. The specific sequences bound by the CDXA protein in the genomic fragments isolated, were determined by DNase I footprinting. From the footprinted sequences, the CDXA consensus binding site was determined. The CDXA protein binds the consensus sequence A, A/T, T, A/T, A, T, A/G. The CAUDAL binding site in the ftz promoter is also included in this consensus sequence. When tested, some of the genomic target sequences were capable of enhancing the transcriptional activity of reporter plasmids when introduced into CDXA expressing cells. This study determined the DNA sequence specificity of the CDXA protein and it also shows that this protein can further activate transcription in cells in culture.
The role of the Drosophila lamin protein in nuclear envelope assembly was studied using a Drosophila in vitro assembly system that reconstitutes nuclei from added sperm chromatin or naked DNA. Upon incubation of the embryonic assembly extract with anti-Drosophila lamin antibodies, the attachment of nuclear membrane vesicles to chromatin surface and nuclear envelope formation did not occur. Lamina assembly and nuclear membrane vesicles attachment to the chromatin were inhibited only when the activity of the 75-kD lamin isoform was inhibited in both soluble and membrane-vesicles fractions. Incubation of decondensed sperm chromatin with an extract that was depleted of nuclear membranes revealed the presence of lamin molecules on the chromatin periphery. In addition, high concentrations of bacterially expressed lamin molecules added to the extract, were able to associate with the chromatin periphery, and did not inhibit nuclear envelope assembly. After nuclear reconstitution, a fraction of the lamin pool was converted into the typical 74- and 76-kD isoforms. Together, these data strongly support an essential role of the lamina in nuclear envelope assembly.
CHox-cad is a chicken homeobox gene whose homeodomain is homologous to the Drosophila caudal and the murine Cdx1 genes. Based on sequence analysis of a 2.5 kb CHox-cad cDNA clone, we deduced that the primary translation product consists of 248 amino acids. Comparison between the cDNA and genomic clones revealed the presence of an intron within the CHox-cad homeodomain between amino acids 44 and 45. The onset of CHox-cad transcription correlates temporarily with the beginning of gastrulation. During primitive streak stages CHox-cad exhibits a caudally localized pattern of expression restricted to the epiblast and the primitive streak. At these stages, CHox-cad transcripts can also be detected in the definitive endoderm cells. Later in embryogenesis CHox-cad is expressed in the epithelial lining of the embryonic gut and yolk sac. After four days of chicken development, no CHox-cad transcripts could be detected. The early CHox-cad posterior expression in the germ layer undergoing gastrulation and its continuous expression in the early endodermal lineage raise the possibility of CHox-cad involvement in the establishment of the definitive endoderm.
CHox E is a novel chicken homeogene that belongs to the H2.0 family of homeodomains. Its homeobox sequence is interrupted by an intron between amino acids 44 and 45. Expression of CHox E during embryogenesis is localized to the central nervous system. The anterior boundary of CHox E expression can initially be localized to rhombomere number 1, later in development this boundary reaches up to the rhombencephalic isthmus. CHox E expression in the spinal cord localizes dorso-ventrally to the dorsal half of the basal plate. CHox E expression is always restricted to the proliferating region, the ventricular zone. As the ventricular zone becomes restricted laterally, so does the CHox E expressing region. Once this region of the ventricular zone ceases to exist, CHox E specific transcripts become undetectable. The site and time of CHox E expression suggest a very early function in the differentiation of the cells derived from that region of the ventricular zone.
Activity of the cat gene driven by the cauliflower mosaic virus 35S promoter has been assayed by transfecting petunia protoplasts with the pUC8CaMVCAT plasmid. In vitro methylation of this plasmid with M.HpaII (methylates C in CCGG sites) and M.HhaI (methylates GCGC sites) did not affect bacterial chloramphenicol acetyltransferase (CAT) activity. It should be noted, however, that no HpaII or HhaI sites are present in the promoter sequence. In contrast, in vitro methylation of the plasmid with the spiroplasma methylase M.SssI, which methylates all CpG sites, resulted in complete inhibition of CAT activity. The promoter sequence contains 16 CpG sites and 13 CpNpG sites that are known to be methylation sites in plant DNA. In the light of this fact, and considering the results of the experiments presented here, we conclude that methylation at all CpG sites leaving CpNpG sites unmethylated is sufficient to block gene activity in a plant cell. Methylation of CpNpG sites in plant cells may, therefore, play a role other than gene silencing.
A chick genomic clone that reveals a high degree of homology to the mammalian and Xenopus bFGF gene has been isolated. The pattern of expression of bFGF has been examined during early chick embryogenesis. RNA blot analysis revealed that chick bFGF is already transcribed at pregastrula stages. Immunolabeling analysis indicated that bFGF protein is present at these early developmental stages and is distributed evenly in the epiblast, hypoblast and marginal zone of the chick blastula. Substances that can inhibit FGF action were applied to early chick blastoderms grown in vitro under defined culture conditions (DCM). Both heparin and suramin were capable of blocking the formation of mesodermal structures in a dose-dependent manner. Our results indicate that FGF-like substances may need to be present for axial structures to develop although they may be acting earlier during the induction of non-axial mesoderm.
We have recently identified and characterized a 53-kDa inner nuclear membrane-associated protein in Drosophila and termed it otefin. Here we report the isolation and characterization of cDNA and genomic clones of the otefin gene. Based on sequence analysis, we deduced that the primary translation product has a calculated mass of 45 kDa, contains many serine and threonine residues, and is mostly hydrophilic. However, in the carboxyl terminus, there is a hydrophobic region which may serve as a membrane anchoring domain. RNA blot analysis indicated that the otefin gene codes for a single poly(A+) transcript of 1.6 kilobases and that relatively large amounts of this transcript are present during developmental stages in which many nuclear divisions occur. Polyclonal antibodies raised against the cDNA translation product react with a 58-kDa mammalian nuclear envelope protein, demonstrating evolutionary conservation.
Southern blots of genomic DNA from a variety of species digested by restriction endonucleases having a four-bp specificity, were probed with a bovine genomic clone consisting of seven tandem poly-TG stretches separated by a 29bp linker sequence. Highly variable DNA 'fingerprint' patterns were obtained in chicken, sheep, and horse, moderately variable DNA 'fingerprints' in mouse and man, and a monomorphic pattern in Drosophila. In chicken, horse and man a (TG)10 synthetic oligonucleotide probe gave results identical to those given by the bovine probe. Furthermore, in chicken the DNA fingerprint variation showed typical Mendelian inheritance and differed from the fingerprints obtained with Jeffreys 33.6 and M13 minisatellite probes. Thus, for a variety of vertebrate species, poly-TG-containing probes can uncover useful genetic variation.
A complete nucleotide sequence of a 4.2-kb genomic fragment containing the Drosophila lamin gene and flanking sequences is presented. Primer extension experiments and sequence analysis revealed that transcription starts from a single promoter. The lamin maternal 2.8-kb transcript and the 3.0-kb zygotic transcript are generated from two alternative polyadenylation sites. The gene contains four exons. The first intron is 7 bp upstream of the first AUG site. The two other introns are located within the alpha-helical rod domain of the protein: one in coil 1B in the 42-amino-acid domain that is absent in vertebrate cytoplasmic intermediate filament proteins and the other in coil 2 at a position different from intron positions within the vertebrate intermediate filament genes. Together with the sequence homology analysis, the data suggest either that the lamin gene was the ancestral gene of intermediate filament genes or that the lamin gene diverged from other intermediate filament genes early in evolution.
Screening of a bovine genomic library with the human minisatellite 33.6 probe uncovered a family of clones that, when used to probe Southern blots of bovine genomic DNA digested with the restriction enzyme HaeIII or MboI, revealed sexually dimorphic, but otherwise virtually monomorphic, patterns among the larger DNA fragments to which they hybridized. Characterization of one of these clones revealed that it contains different minisatellite sequences. The sexual dimorphism hybridization pattern observed with this clone was found to be due to multiple copies of two tandemly interspersed repeats: the simple sequence (TG)n and a previously undescribed 29-bp sequence. Both repeats appear to share many genomic loci including autosomal loci. In contrast, Southern analysis of AluI- or HinfI-digested bovine DNA with the (TG)n repeat used as a probe yielded substantial polymorphism. These results show that (i) different minisatellites can be found in a cluster, (ii) both simple and more complex repeated sequences other than the simple quaternary (GATA)n repeat can be sexually dimorphic, and (iii) simple repeats can reveal substantial polymorphism.
Several Drosophila genes involved in the control of segmentation and segment identity share a 183-bp conserved sequence termed homeo box. Homeo box sequences have been detected and cloned from the genomes of insects like Drosophila to vertebrates such as mouse and man. Two chicken homeo box genes CHox1 and CHox3, are described. Cloning of the CHox1 and CHox3 homeo boxes was performed using Drosophila and murine homeo box sequences as probes under low-stringency conditions. Analysis of both chicken homeo box sequences revealed them to be homeo boxes that have diverged from the Antennapedia class with homologies to homeo boxes of other organisms in the range of 75-42% at the nucleotide level and 69-41% at the protein level. Analysis of CHox3 expression during early embryo development showed that the gene codes for five transcripts 1.3, 1.9, 2.6, 5.6 and 7.9 kb in size. Three of the transcripts (1.3, 1.9 and 5.6 kb) are also recognized by a flanking non-homeo box containing probe. The levels of the different transcripts changed during the first five days of development. The most abundant transcripts (1.3 and 1.9 kb) are already present at the time the egg is laid. Their transcription peaks at day 1 of incubation and then decreases. The CHox1 transcripts are present at very low levels between days 2.5 and 4 of development. These two chicken genes represent bona fide Hox genes in a branch of vertebrates that evolved parallel to mammals.
Chicken sperm chromatin initiated an assembly of interphase-like nuclei in a cell-free cytoplasmic preparation from 1-6 h old Drosophila melanogaster embryos. The formation of these interphase-like nuclei from the condensed sperm chromatin happened in a series of distinct steps. Anti-Drosophila lamin monoclonal antibody stained the assembled nuclei in a pattern indistinguishable from normal Drosophila nuclei. This assembly process required an ATP regenerating system and could be blocked by the addition of novobiocin into the cell-free extract.