Electron microscopy and restriction enzyme mapping reveal additional intervening sequences in the chicken ovalbumin split gene

The Eco RI fragment “b” of chicken DNA (Breathnach, Mandel and Chambon, 1977), which contains the sequences coding for the 5′ quarter of ovalbumin mRNA (ov mRNA), has been isolated by molecular cloning using a “shotgun” approach. Electron microscopy and restriction enzyme analysis have revealed that the sequences coding for the 5′ quarter (～500 nucleotides) of ov mRNA are split into four regions separated by three intervening sequences. The cloning procedure seems to be reliable, since the restriction enzyme pattern of the cloned Eco RI fragment “b” is similar to that of the corresponding chromosomal DNA fragment. There is no evidence supporting the existence of a 150–200 nucleotide long sequence at the 5′ end of the ov mRNA similar to the “leader” sequences found at the 5′ end of some adenovirus and SV40 mRNAs.     

Molecular structure and flanking nucleotide sequences of the natural chicken ovomucoid gene

Five independent clones containing the natural chicken ovomucoid gene have been isolated from a chicken gene library. One of these clones, CL21, contains the complete ovomucoid gene and includes more than 3 kb of DNA sequences flanking both termini of the gene. Restriction endonuclease mapping, electron microscopy and direct DNA sequencing analyses of this clone have revealed that the ovomucoid gene is 5.6 kb long and codes for a messenger RNA of 821 nucleotides. The structural gene sequence coding Ifor the mature messenger RNA is split into at least eight segments by a minimum of seven intervening sequences of various sizes. The shortest structural gene segment is only 20 nucleotides long. All seven intervening sequences are located within the peptide coding region of the gene, and the sequences at the 5' and 3' untranslated regions of the mRNA are not interrupted by intervening sequences. The DNA sequences of the regions flanking the 5' and 3' termini of the gene have been determined. Thirty nucleotides before the start of the messenger RNA coding sequence is the heptanucleotide TATATAT, which is also present in a similar location relative to the chicken ovalbumin gene and other unique sequence eucaryotic genes. This sequence resembles that of the Pribnow box in procaryotic genes where a promoter function has been implicated. Seven nucleotides past the 3' end of the gene is the tetranucleotide TTGT, a sequence found to be present at identical locations as either TTTT or TTGT in other eucaryotic genes that have been sequenced. These conserved DNA sequences flanking eucaryotic genes may serve some regulator function in the expression of these genes.     

The ovalbumin split gene: molecular cloning of Eco RI fragments "c" and "d".

The Eco RI fragments "c" and "d" of the ovalbumin gene (1, 2) have been isolated by molecular cloning. Restriction enzyme mapping and electron microscopy have confirmed that the two fragments contain the same ovalbumin mRNA coding sequences. These sequences are split into two regions which have been mapped in fragments "c" and "d". There is no evidence that the ovalbumin mRNA sequences contained in these fragments could be further interrupted. Our results confirm that the presence of Eco RI fragment "d" in some chickens is due to the existence of an allelic variant of the ovalbumin gene which contains an additional Eco RI site within the region corresponding to Eco RI fragment "c". This additional Eco RI site appears to be the main difference between the two alleles. Finally, our results provide a direct demonstration that most of the ovalbumin mRNA sequences are encoded for by Eco RI fragments "a", "b" and "c".     

The DNA sequence of Bombyx mori fibroin gene including the 5' flanking, mRNA coding, entire intervening and fibroin protein coding regions.

Sequence analysis of the cloned genomic fibroin gene and cDNA containing the sequence complementary to fibroin mRNA has been carried out for the regions covering the 5′ flanking, mRNA coding, entire intervening sequence and its borders, and fibroin coding sequences. The sequences determined on the gene extend from nucleotide ?552 to +1497 (assigning +1 to the cap locus); sequence analysis of the cDNA has confirmed our previous mapping of the cap locus (Tsujimoto and Suzuki, 1979). Comparison of the nucleotide sequence of the genomic gene with that of cDNA has confirmed the existence of an intervening sequence 970 bases long. The sequence comparison also pinpointed the 5′ coding-intervening junction at +64?66 and the 3′ intervening-coding junction at +1034?1036. Both the 5′ and 3′ junctions of the fibroin gene (insect) share homologous segments of about 10 nucleotides each with the published sequences of β-globin (mammal), immunoglobulin (mammal) and ovalbumin (avian) genes. A long inverted repeat sequence (17 of 23 base match) has been found next to the junctions within the intervening sequence of the fibroin gene. The repetitious sequence that codes for the Gly-Ala peptide characteristic of fibroin protein begins at position +1448. The characteristics of the N terminal portion of fibroin protein (or its precursor) are discussed, as are the features of the 5′ flanking sequence of the gene and the mRNA sequence (with special attention to the putative promoter sequence for the gene), the possible secondary structure and a sequence complementary to the 3′ end of 18S ribosomal RNA at the 5′ proximal region of fibroin mRNA.     

Mutations in putative intervening sequences of the mitochondrial cytochrome b gene of yeast produce abnormal cytochrome b polypeptides.

The apoprotein of yeast cytochrome b is translated on mitochondrial ribosomes and coded for by a split gene which is located in the COB-BOX region on mitochondrial DNA. With the aid of an antibody against cytochrome b, we identified the cytochrome b-cross-reacting polypeptides of respiration-deficient mutants mapping either in coding or intervening sequences of the cytochrome b gene. Most mutations in the coding regions caused the accumulation of a single apocytochrome b fragment whose apparent molecular weight (12,000 to 26,600) depended on the map position of the mutation. In contrast, mutations in putative intervening sequences often led to multiple new polypeptides immunologically related to apocytochrome b. Some of these abnormal polypeptides were considerably larger than wild type apocytochrome b. This suggests that mutations in intervening sequences can thus generate aberrant polypeptide products.     

Structure of the chicken apo very low density lipoprotein II gene

We describe two cloned genomic DNA fragments, both bearing the entire apo very low density lipoprotein II gene. Electron microscopy and restriction enzyme mapping showed that this gene is split into at least four coding sequences by three or more intervening sequences. A very short exon at the 5'-end of the gene is separated by a 1.5-kilobase intron from the second exon, which codes for the AUG initiation codon of the mRNA.     

Effect of estrogen on ovalbumin gene expression in differentiated nontarget tissues

By use of cloned DNA fragments as probes, low levels of ovalbumin RNA sequences (structural and intervening sequences) were detected in nuclear RNA extracts of nontarget tissues, such as liver, spleen, brain, and heart of chicks. The expression of the ovalbumin gene sequences was hormone dependent. In estrogen-stimulated chicks, a low level of ovalbumin RNA sequences, ranging from 0.2 to 0.7 molecule per cell, was present in nontarget tissues while less than 0.01 molecule per cell could be found in the same tissues of unstimulated chicks. A significant amount of the ovalbumin mRNA sequences was also found in polysomes of liver and brain. The ovalbumin mRNA sequences could be translated into proteins which were only localized in a few cells among the entire population of liver cells as determined by an immunocytochemical assay. These results suggest that there are some cells in liver, spleen, heart, and brain which can respond to hormone stimulation and produce ovalbumin mRNA and its translational product.     

Structure of the rat prolactin gene

The organization and sequence of the rat preprolactin gene has been investigated. Analysis of two different plasmids containing pituitary cDNA inserts has provided the complete 681-nucleotide coding sequence of preprolactin as well as 17 nucleotides preceding the initiation codon and 90 nucleotides following the termination codon. Digestion of rat chromosomal DNA with the restriction endonuclease Eco RI followed by size fractionation and hybridization to a labeled prolactin cDNA probe has demonstrated that prolactin genomic sequences are located on 6.0-, 3.9-, and 2.9-kilobase fragments. The 6.0- and 3.9-kilobase fragments were isolated from a library of cloned rat DNA fragments. The sequence of more than 1800 nucleotides of the cloned DNA has been determined. The sequenced region contains coding regions of 180 and 189 nucleotides which specify the COOH-terminal 123 amino acids of the 227-amino-acid sequence of rat preprolactin. These coding regions are separated by an intervening sequence of 597 nucleotides. At least one other large intervening sequence separates this region from the region coding for the NH2-terminal portion of preprolactin. Hybridization experiments suggested that the intervening sequences of the rat prolactin gene contain DNA sequences which are repeated elsewhere in the rat genome.     

The presence of ovalbumin mRNA coding sequences in multiple restriction fragments of chicken DNA.

Chicken DNA has been digested with restriction enzymes and the size distribution of the DNA fragments containing ovalbumin specific sequences has been examined after separation of the fragments on agarose gels and transfer to nitrocellulose sheets. Hybridisation with terminally 32P-labelled ovalbumin mRNA fragments or with RNA populations transcribed from the DNA of a hybrid plasmid containing ovalbumin sequences was used to locate the DNA fragments coding for ovalbumin. Digestion with enzymes which do not cut within the portion of the ovalbumin gene synthesised from ovalbumin messenger RNA in vitro has shown the presence of several defined fragments carrying ovalbumin specific sequences. Possible explanations of these observations are discussed.     

The sequence of a human immunoglobulin epsilon heavy chain constant region gene, and evidence for three non-allelic genes

An immunoglobulin epsilon heavy chain gene was isolated from a DNA library of the human epsilon chain-producing myeloma 266B1 , using a JH gene region probe. The gene was shown to be the one expressed in the myeloma by Southern hybridisation analysis and by comparison of nucleotide sequences with the known amino acid sequence of the epsilon chain made by the myeloma. The gene consists of a variable region segment separated from a constant region segment by a 3.5-kb intervening sequence. The complete sequence of the constant region gene segment shows that this segment is split by intervening sequences into four coding segments corresponding to the four constant region domains of the protein. Using the cloned epsilon constant region gene segment as a probe we obtained evidence, from Southern hybridisation analysis, for three non-allelic epsilon constant region genes. An order on the chromosome for these three genes can be predicted from their pattern of retention in myeloma 266B1 DNA.     

PO149, a new member of pollen pectate lyase-like gene family from alfalfa

PO149 is a low-copy-number gene expressed in the late stages of pollen development. The promoter region contains no similarities in DNA sequence to those of other pollen-specific genes, except for a tobacco sequence (AAATGA), which occurs four times in this alfalfa gene and much further upstream than in tobacco. Four distinct TATA boxes were detected in the promoter with the distal and proximal TATA boxes being separated by a spacer of 269 nucleotides. Hairpin loop structures were found in the 5-and 3-untranslated regions of PO149 mRNA. The coding region of PO149 is interrupted by two introns and encodes a putative prepeptide of 450 amino acids with homology to pollen pectate lyase-like proteins and pollen allergens. The coding region also contains sequences characteristic of both a signal peptide and a nuclear localization signal.     

Plant nuclear tRNAMet genes are ubiquitously interrupted by introns

We have isolated three independent clones for nuclear elongator tRNA^Met genes from an Arabidopsis DNA library using a tRNA^Met-specific probe generated by PCR. Each of the coding sequences for tRNA^Met in these clones is identical and is interrupted by an identical 11 bp long intervening sequence at the same position in the anticodon loop of the tRNA. Their sequences differ at two positions from the intron in a soybean counterpart. Southern analysis of Arabidopsis DNA demonstrates that a gene family coding for tRNA^Met is dispersed at at least eight loci in the genome. The unspliced precursor tRNA^Met intermediate was detected by RNA analysis using an oligonucleotide probe complementary to the putative intron sequence. In order to know whether introns commonly interrupt plant tRNA^Met genes, their coding sequences were PCR-amplified from the DNAs of eight phylogenetically separate plant species. All 53 sequences determined contain 10 to 13 bp long intervening sequences, always positioned one base downstream from the anticodon. They can all be potentially folded into the secondary structure characteristic for plant intron-containing precursor tRNAs. Surprisingly, GC residues are always present at the 5-distal end of each intron.