Molecular evolution of the globin gene cluster E in two distantly related midges, Chironomus pallidivittatus and C. thummi thummi.

We have studied the evolutionary dynamics of a cluster of insect globin genes by comparing the organization and sequence of the gene group in two distantly related species, Chironomus pallidivittatus and C. t. thummi. Although the general architecture of the globin gene cluster has been conserved, we have found an additional, previously undescribed gene (named Cpa F) in C. pallidivittatus which shows signs of accelerated sequence evolution at nonsynonymous codon positions. This new gene is clearly functional, as demonstrated by Northern analysis. Comparison of paralogous and orthologous genes reveals patterns of intraspecific sequence homogenization. The head-to-head-oriented globin 3 and 4 gene pairs in C. t. thummi and the gb 4 gene pair in C. pallidivittatus have been efficiently homogenized, probably by gene conversion, in their promoter and coding regions. Inverted transcriptional orientation seems to favor efficient conversion. The orthologous genes from C. t. thummi and C. pallidivittatus reveal different levels of sequence conservation, ranging from 85.3 to 94.7% amino acid identity. Surprisingly, globin gene E, for which up to now no corresponding protein has been detected in the larval hemolymph of C. t. thummi, shows the highest degree of interspecies sequence conservation. This points to an essential, as yet unknown function of this globin. The usefulness of globin gene comparisons for dating speciation events in Chironomus is discussed.

Structure and erythroid cell-restricted expression of a chicken cDNA encoding a novel zinc finger protein of the Cys + His class.

We report the cloning, sequence analysis and expression pattern of chGfi, a zinc finger protein (Zfp)-encoding cDNA that was isolated from a cDNA library constructed with RNA from avian erythroblastosis virus (AEV)-transformed primary chicken erythroblasts. The 1387-bp-long chGfi cDNA encodes a full-length 337-amino-acid (aa) protein that contains six zinc fingers (Zf) of the 2Cys + 2His class at its C-terminus. Immunoblotting experiments with extracts from bone marrow cells detected a 38-kDa protein that corresponds to the M(r) of 38,690 calculated for the protein deduced from chGfi. The chGfi protein is most homologous to the rat Gfi-1 showing a sequence similarity of 92% over the Zf region and only two exchanges within the N-terminal 19 aa that constitute the Gfi-1 repressor domain. Expression of chGfi is only detected in transformed primary erythroblasts, in erythroid cells of the primitive and definitive lineage and in bone marrow cells. chGfi activity does not vary significantly during differentiation of transformed primary erythroblasts of the definitive lineage. No chGfi expression is detected in cells of the myeloid and lymphoid lineages or in a total of nine different organs of adult origin. Our results indicate that chGfi expression is restricted to erythroid cells of the primitive and definitive lineage.

A caudal-type homeobox gene activity associated with the development and regeneration of the liver.

Indirect evidence suggests that a chicken homologue to caudal, CHox-cad, might be involved in the development of endoderm-derived tissues and in the regeneration of the adult intestinal epithelium. Using the adult liver as an experimental system, we have investigated whether CHox-cad expression can be induced by a regenerative stimulus following partial hepatectomy. Under conditions of normal growth, CHox-cad is expressed in the embryonic but not in the adult liver. In the regenerating adult liver, immunohistochemical analyses and in situ hybridization experiments revealed a varying CHox-cad expression pattern extending from 6 h to 11 days posthepatectomy. Early in regeneration, 6 h and 18 h after partial hepatectomy, CHox-cad expression is confined to hepatocytes present in the periphery of the lobuli and to putative hepatic progenitor cells in the portal spaces. At later stages, from 30 h posthepatectomy onwards, CHox-cad activity is detected predominantly in cells of the hepatic progenitor cell compartment, the portal spaces. By day 8, CHox-cad-expressing cells have occupied marginal positions in some portal spaces and appear to penetrate into the periportal parenchyma. With progressing regeneration CHox-cad activity gradually declines reaching low levels in cells of a few portal spaces by day 11 post hepatectomy. As expected for a putative transcription factor, CHox-cad protein is localized to the cell nucleus. Immunodetection of PCNA, a marker of cell proliferation, revealed a much more widespread distribution of mitotic cells as compared to CHox-cad-expressing cells. In the region of overlapping expression domains, coexpression of CHox-cad and PCNA in the same cells was rare in most instances. There is evidence to suggest that CHox-cad is involved in the differentiation of liver progenitor cells to mature hepatocytes. The similarity of the CHox-cad expression pattern in the early developing liver and in the adult regenerating liver may indicate the reactivation of a genetic program operative during early stages of liver morphogenesis.

Differential expression of the chicken Pax-1 and Pax-9 gene: in situ hybridization and immunohistochemical analysis.

We report the cloning, partial sequence analysis, and spatiotemporal expression of the chicken Pax-1 (chPax-1) and Pax-9 (chPax-9) gene, two closely related members of the paired box-containing (PAX) gene family. The chPax-1 gene encodes RNAs of 2.0 and 4.3 kb and a 42 kD protein while the gene products of chPax-9 are represented by 1.9 and 3.1 kb transcripts and a 39 kD protein. In situ hybridization and immunohistochemical analyses reveal chPax-1 expression in the developing pectoral girdle, in cells of the ventral part of sclerotomes, in sclerotome cells of the perichordal tube, and, later in development, in sclerotome-derived cells of the intervertebral disks. Other chPax-1 expression domains detected in the mesenchyme surrounding the atlas and axis and in chondrocytes of immature vertebral bodies, so far unreported for mouse Pax-1, correlate with as yet unexplained malformations in the mouse Pax-1 mutant undulated and Undulated-short tail. Overlapping expression of chPax-1 and chPax-9 is detected in epithelial cells of the embryonic and adult thymus and in cells of the developing intervertebral disks. Unlike chPax-1, however, chPax-9 is not expressed in those perichordal sclerotome cells which are thought to give rise to vertebral bodies. Furthermore, chPax-9 gene products are detected in circumscribed areas of mesenchyme in the metatarsus and in entodermal derivatives, i.e., in the lining epithelium of the developing pharynx and of the embryonic and adult esophagus.

Localization of deoxyribonuclease I gene transcripts and protein in rat tissues and its correlation with apoptotic cell elimination.

The expression and distribution of deoxyribonuclease I (DNase I) in rat parotid gland, kidney, small intestine and keratinized epithelium was further analysed at the level of its mRNA by in situ hybridization and correlated to immunohistochemical results using polyclonal anti-DNase I antibodies. High amounts of DNase I-specific mRNA and immunoreactivity were detected in the parotid gland, kidney and small intestine in agreement with previous immunohistochemical results. In the parotid gland, both the DNase I-specific mRNA and antigenicity were detected within the secretory cells. In the kidney, DNase I gene transcripts were localized in distal tubules and the collecting duct system. In this organ an identical localization of DNase I antigenicity was obtained. In the small intestine only the enterocytes covering the villi were shown to express DNase I-specific mRNA; the highest level being detected within the enterocytes along the lower third of the villi. In contrast, the highest level of immunoreactivity was found in enterocytes covering the middle and upper thirds of the villi. Within the stratified epithelium of the tongue, DNase I gene transcription and protein expression started in lower parts of the stratum spinosum and reached into the stratum granulosum. However, the gradient of DNase I gene transcript expression appeared to be shifted to lower layers of the stratum spinosum when compared to DNase I immunoreactivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and structure of a chicken zinc finger cDNA: restricted expression in developing neural crest cells.

The cloning, DNA sequence analysis and expression pattern of a chicken cDNA, cKr2, is described. cKr2 is a 4492-bp cDNA that encodes a 1173-amino-acid (aa) protein with two domains: the N-terminal portion contains 16 zinc fingers (Zf) of the 2Cys + 2His class, while the C-terminal domain contains a stretch of 181 aa which consists of seven consecutive sequence repeats each being 24 aa in length. The aa sequence repeats harbor a putative DNA-binding helix-turn-helix motif. Northern and Western blotting experiments indicate cKr2 expression from days 2 to 12 of development. In situ hybridization and immunohistochemical analyses using an anti-cKr2 antibody and the HNK-1 antibody, a marker of neural crest cells, revealed cKr2 activity in cephalic and trunk neural crest-derived cells. cKr2 is expressed predominantly in differentiating Schwann cells lining the nerves which innervate the branchial arches and limb buds, in cells of the sympathetic ganglia and aortic plexus, and in putative neuroblasts of dorsal root ganglia. The nuclear localization of the cKr2-encoded protein is consistent with its presumed role as a transcription factor.

Continued expression of the chicken caudal homologue in endodermally derived organs.

We show that the CHox-cad gene is continuously expressed throughout embryogenesis from Days 1 to 14 of development. In the adult chicken, CHox-cad appears to be expressed exclusively in the gut. Genomic Southern blot hybridization experiments suggest that there may be only a single CHox-cad gene per haploid genome from which RNAs of 2.9 and 1.6 kb are transcribed. In situ hybridization to sections of 80-hr to 12-day chick embryos with a CHox-cad probe reveals that this gene is expressed at high levels in organs of endodermal origin: the developing liver, pancreas, lung, and in the epithelial lining of the entire intestine. By Day 16 of development, CHox-cad signals are observed in the epithelial lining of the gut but no longer in the other endodermally derived organs. The coincidence of CHox-cad expression with the onset of development of these endoderm-derived organs may indicate an important role for that gene during the early steps of organogenesis.

Primary structure and expression of a chicken cDNA encoding a protein with zinc-finger motifs.

We report the cloning and sequence analysis of a chicken nearly full-length cDNA clone, cKr1, encoding a protein of 509 amino acids which contains ten 2Cys + 2His-type zinc-finger motifs arranged in two separate sets of five zinc fingers each. The cKr1 transcripts are detected in organs of the adult chicken, predominantly in the brain and lung. At day 4 of embryonic development strong cKr1 hybridization signals are found in the brain and neural tube, in the mesonephros and in the gut, respectively.

Genomic organization and primary structure of five homologous pairs of intron-less genes encoding secretory globins from the insect Chironomus thummi thummi.

From a Chironomus thummi thummi genomic library we have isolated two distinct recombinant phages, CttG-1 and CttG-3, each carrying a cluster of five homologous globin genes. In addition to the previously reported nucleotide sequence of globin gene D (Antoine and Niessing, 1984) we present the chromosomal arrangement, primary structure and predicted amino acid sequence of nine globin genes. The divergently transcribed globin genes all lack introns, they encode secretory preglobins each containing a highly conserved signal peptide. The amino acid sequences deduced from the globin genes correspond to globin III and variants thereof, to globin IV, and to a novel globin, whose direct amino acid sequence has not yet been reported.

Chromosomal arrangement of the duck alpha-globin genes and primary structure of the embryonic alpha-globin gene pi.

A recombinant lambda Charon 4A bacteriophage, D alpha G-1, carrying the genes coding for the duck embryonic (pi') and adult (alpha A, alpha D) alpha-like globins was isolated from a previously constructed duck DNA recombinant library. The three globin genes are transcribed from the same DNA strand and are arranged in the order of their expression during development: 5'-pi'-alpha D-alpha A-3'. We have determined the complete nucleotide sequence of the duck pi'-globin gene, including the flanking regions. Due to the unusual length of intron 1 (963 bp) and intron 2 (568 bp) the 2167-bp duck pi'-globin gene is by far the largest among all known mammalian or avian alpha- and beta-globin genes. For instance, the duck pi'-globin gene introns are almost twice as long as those of the chicken pi'-globin genes. A surprisingly high degree of nucleotide sequence homology (88%) has been found for the 5' flanking region (positions -1 to -223) of the duck and chicken pi'-globin gene.

Yeast is unable to excise foreign intervening sequences from hybrid gene transcripts.

To investigate whether transcripts from foreign split genes are correctly processed in yeast cells we have constructed two hybrid genes by inserting into the split yeast actin gene an intron-containing fragment from either the Acanthamoeba actin I gene or the duck alpha D-globin gene. The hybrid genes were inserted into the autonomously replicating yeast plasmid YRp7, which was then used to transform yeast cells. It was found that the yeast but not the foreign intervening sequences were excised from the chimeric transcripts. This indicates that the recognition of intervening sequences or the splicing mechanism of RNA polymerase II transcripts is not universal.

The primary structure of the duck alpha D-globin gene: an unusual 5' splice junction sequence.

The complete nucleotide sequence of the duck minor alpha D-globin gene including the flanking regions has been determined. A unique structural feature of the alpha D-globin gene is a GC instead of the invariant GT dinucleotide at the 5' end of the second intervening sequence. The 1013 base pair long gene has otherwise all the characteristics normally attributed to a functional globin gene. Indirect evidence suggests that the alpha D-globin gene is expressed in vivo.

The complete nucleotide sequence of the duck alpha A-globin gene.

We report the entire nucleotide sequence of the duck alpha A-globin gene including 330 nucleotides of the 5'-flanking region. The amino acid coding sequence of the 815-bp-long gene is interrupted at preserved locations by two intervening sequences. In contrast to all other vertebrate globin genes investigated so far, the first intervening sequence (150 bp) of the duck alpha A-globin gene exceeds the size of the second intervening sequence (104 bp) by 46 bp. Moreover, the alpha A-globin gene has an extraordinary GC-rich 5'-flanking region. Conserved regions are identified which might encode signals for transcriptional initiation, RNA splicing and poly(A) addition.

The isolation and partial characterization of linked alpha A- and alpha D-globin genes from a duck DNA recombinant library.

Genomic DNA from an adult duck (Cairina moschata) was used to construct a library of cloned DNA fragments in the vector lambda Charon 4A. Screening of the DNA library resulted in the isolation of a recombinant, D alpha G-1, which carries both the adult duck alpha A- and alpha D-globin genes. The two globin genes are separated by approx. 2.2 kb of DNA, they are encoded by the same DNA strand and their orientation with respect to the direction of transcription is 5'- alpha D- alpha A-3'. Partial sequence analyses indicate that the two alpha-globin genes contain intervening sequences at positions homologous to those in chicken and mammalian alpha-globin genes.

Globin messenger precursor RNA in duck immature red blood cells.

A procedure is described for the chromatographic analysis of RNA under fully denaturing conditions on cross-linked Sepharose. Chromatography of DNA . RNA hybrids, poly(C) . poly(G) hybrids and complexes of poly(C) . hnRNA on Sepharose CL in pure formamide at 46 degrees C leads to denaturation and strand separation of the hybrid structures. Using this procedure, nuclear RNA from duck immature red blood cells was resolved according to molecular size and assayed for the presence of globin mRNA sequences by hybridization with complementary DNA. Two size classes of putative globin mRNA precursor molecules were detected at an elution position corresponding to 14--18 S and 23--28 S. As judged from chromatographic analysis on poly(U)-Sepharose, about 70% of the 14--18-S globin precursor RNA is polyadenylated while only 11% of the putative 23--28-S precursor RNA has a poly(A) tract. Inhibition of transcription by actinomycin D and pulse-chase experiments indicate a half-life of less than 7.5 min for these precursor RNA species.