Cloned embryonic DNA sequences flanking the mouse immunoglobulin C gamma 3 and C gamma 1 genes

To investigate the DNA surrounding genes for immunoglobulin heavy chain constant (CH) regions, we have isolated two clones bearing a C gamma 3 gene and two bearing a C gamma 1 gene from a library of mouse embryo DNA fragments. The C gamma 3 clones span 8.6 kilobase pairs (kb) on the 5' side of the gene and 6.7 kb on its 3' side, while the C gamma 1 clones together span 13 kb of 5' flanking sequence and 2.5 kb of 3' flanking sequence. Restriction mapping of the C gamma 3 gene indicates that intervening sequences divide the gene into segments of domain size, as in other CH genes. Hybridization of clone fragments to restriction digests of mouse DNA indicates that both the C gamma 1 and C gamma 3 genes probably occur as single copies in the genome. Moreover, the entire cloned sequences on the 5' side of both genes appear to be unique in the genome, indicating that no large common sequences flank CH genes. Restriction data suggest that the C gamma 3 gene is 37-40 kb 5' to the C gamma 1 gene.     

Identification of EcoRV fragments spanning the N alpha-tubulin gene of Physarum

The N alpha-tubulin gene of Physarum polycephalum has an EcoRV site at codons 252/253. EcoRV digestion of physarum DNA generated two EcoRV fragments per gene copy comprising both coding and flanking sequences. Hybridisation probes which included coding sequences upstream from the central EcoRV site cross-hybridised with another alpha-tubulin gene. Probes derived from either 5'- or 3'-flanking regions were gene-specific. These probes identified two EcoRV fragments in the haploid strain CLdAXE viz 5.4 kb (5'-fragment) and 6.2 kb (3'-fragment). The same two fragments were identified in EcoRV digests of DNA of the diploid strain M3CVIII, and a second form of the gene was also identified comprising two fragments viz 5.0 kb (5'-end) and 5.5 kb (3'-end). Both forms gave rise to an identical 4.65 kb HindIII fragment as judged by restriction mapping.     

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.     

Simple DNA sequences in homologous flanking regions near immunoglobulin VH genes: a role in gene interaction?

Five closely related immunoglobulin VH genes (subgroup II) were compared by sequencing of several kb of DNA. In three of the genes homology greater than 75% was found along an area of 4 kb that includes the coding region. The homology in flanking regions is only slightly lower than that in the coding sequences. Two other genes, which are located on the same EcoRI fragment, show high homology to the first three genes in the coding and immediately flanking regions. In more distant flanking regions no homology is found with the first three genes. This indicates that their evolutionary history differs from that of the other three genes. A region of simple DNA sequence composed of repetitive TCC and TCA elements was found at a distance of approximately 380 bp upstream from the initiator ATG of these VH genes. This region is the site where the two sets of genes abruptly start to diverge. The structure of the simple DNA sequence in the various VH genes suggests that it may be involved in gene interaction. We propose that both simple DNA sequences and homology in flanking regions serve a function in the correction of VH genes, which seem to be rather free to diverge and drift into pseudogenes. A correction mechanism may help this gene family to maintain its two major features, multiplicity and diversity.     

Gene-associated CpG islands in plants as revealed by analyses of genomic sequences

We screened plant genome sequences, primarily from rice and Arabidopsis thaliana, for CpG islands, and identified DNA segments rich in CpG dinucleotides within these sequences. These CpG-rich clusters appeared in the analysed sequences as discrete peaks and occurred at the frequencies of one per 4.7 kb in rice and one per 4.0 kb in A. thaliana. In rice and A. thaliana, most of the CpG-rich clusters were associated with genes, which suggests that these clusters are useful landmarks in genome sequences for identifying genes in plants with small genomes. In contrast, in plants with larger genomes, only a few of the clusters were associated with genes. These plant CpG-rich clusters satisfied the criteria used for identifying human CpG islands, which suggests that these CpG clusters may be regarded as plant CpG islands. The position of each island relative to the 5'-end of its associated gene varied considerably. Genes in the analysed sequences were grouped into five classes according to the position of the CpG islands within their associated genes. A large proportion of the genes belonged to one of two classes, in which a CpG island occurred near the 5'-end of the gene or covered the whole gene region. The position of a plant CpG island within its associated gene appeared to be related to the extent of tissue-specific expression of the gene; the CpG islands of most of the widely expressed rice genes occurred near the 5'-end of the genes.     

Human U1 small nuclear RNA genes: extensive conservation of flanking sequences suggests cycles of gene amplification and transposition.

The DNA immediately flanking the 164-base-pair U1 RNA coding region is highly conserved among the approximately 30 human U1 genes. The U1 multigene family also contains many U1 pseudogenes (designated class I) with striking although imperfect flanking homology to the true U1 genes. Using cosmid vectors, we now have cloned, characterized, and partially sequenced three 35-kilobase (kb) regions of the human genome spanning U1 homologies. Two clones contain one true U1 gene each, and the third bears two class I pseudogenes 9 kb apart in the opposite orientation. We show by genomic blotting and by direct DNA sequence determination that the conserved sequences surrounding U1 genes are much more extensive than previously estimated: nearly perfect sequence homology between many true U1 genes extends for at least 24 kb upstream and at least 20 kb downstream from the U1 coding region. In addition, the sequences of the two new pseudogenes provide evidence that class I U1 pseudogenes are more closely related to each other than to true genes. Finally, it is demonstrated elsewhere (Lindgren et al., Mol. Cell. Biol. 5:2190-2196, 1985) that both true U1 genes and class I U1 pseudogenes map to chromosome 1, but in separate clusters located far apart on opposite sides of the centromere. Taken together, these results suggest a model for the evolution of the U1 multigene family. We speculate that the contemporary family of true U1 genes was derived from a more ancient family of U1 genes (now class I U1 pseudogenes) by gene amplification and transposition. Gene amplification provides the simplest explanation for the clustering of both U1 genes and class I pseudogenes and for the conservation of at least 44 kb of DNA flanking the U1 coding region in a large fraction of the 30 true U1 genes.     

Evolution of the Autosomal Chorion Locus in Drosophila. I. General Organization of the Locus and Sequence Comparisons of Genes S15 and S19 in Evolutionarily Distant Species

We have isolated clones corresponding to the autosomal chorion locus of Drosophila melanogaster, from two distantly (D. virilis and D. grimshawi) and one closely (D. subobscura) related species. In all the species the locus is unique within the genome and encompasses the same four chorion genes and an adjacent nonchorion gene, in the same order. In all species the locus specifically amplifies in the ovary, as in D. melanogaster. We present the nucleotide sequences of DNA segments that total 8.3 kb in length and include gene s15-1 from D. subobscura, D. virilis, and D. grimshawi as well as gene s19-1 from D. subobscura and D. grimshawi. They show clearly nonuniform rates of divergence, both within and outside the limits of the genes. Highlighted by a background of extensive sequence divergence elsewhere in the extragenic region, highly conserved elements are observed in the 5' flanking DNA and might represent regulatory elements.     

Organization of ribosomal RNA gene repeats of the mouse.

The organization of the ribosomal RNA (rRNA) genes of the mouse was determined by Southern blot hybridization using cloned rDNA fragments as probes, which could encompass the entire spacer region between two rRNA gene regions. The rRNA genes are organized into tandem repeats of nearly uniform length of about 44 kb. The heterogeneity detected in the nontranscribed spacer appears to be caused by its sequence rather than its length difference. At least three kinds of repetitive sequences are present in the non-transcribed spacer region; two of them are located 13 kb upstream from the 5'-end of 18S RNA gene and the other located 1 to 4 kb downstream from the 3'-end of 28S RNA gene.     

Evolution of a D. melanogaster glutamate tRNA gene cluster

We have determined the DNA sequence of a cloned cluster of essentially identical glutamate tRNA genes of D. melanogaster. The cluster consists of five genes: a gene triplet spanning approximately 0.55 kb followed by a 0.45 kb gene doublet 3.0 kb downstream. The genes are all arranged with the same polarity, do not encode the tRNA CCA end and contain no intervening sequences. Examination of the 5' and 3' sequences immediately flanking each gene reveals a striking pattern of sequence homologies between certain of the genes, which suggests a possible evolutionary history of this gene cluster. We propose that two ancestral genes each gave rise to gene doublets by duplication, while one of these gene pairs then gave rise, in turn, to a trio of genes as a result of unequal crossover.     

The 5.8S RNA gene sequence and the ribosomal repeat of Schizosaccharomyces pombe

We have characterized the rRNA gene repeat in Schizosaccharomyces pombe. This repeat, which does not contain the 5S RNA gene, is found in a 10.4 kb HindIII DNA fragment. We have determined the nucleotide sequences of the S. pombe 5.8S RNA gene and intergenic spacers from two different 10.4 kb DNA fragments. Analysis of isolated total cellular 5.8S RNA revealed the presence of eight species of 5.8S RNA, differing in the number of nucleotides at the 5'-end. The eight 4.8S RNA species vary in length from 158 to 165 nucleotides. Apart from the heterogeneity observed at the 5'-end, the sequence of the eight 5.8S RNA species appears to be identical and is the same sequence as coded for by the 5.8S genes. The gene sequence shows great homology to the 5.8S RNA genes or S. cerevisiae and N. crassa. Most of the base differences are confined to the highly variable stem though to be involved in co-axial helix stacking with the 25S RNA, where base pairing is nearly identical despite the sequence differences. Secondary structure models are examined in light of 5.8S RNA oligonucleotide conservation across species from yeasts to higher eukaryotes.     

Evolutionary constraint in flanking regions of avian genes

An important comprehension from comparative genomic analysis is that sequence conservation beyond neutral expectations is frequently found outside protein-coding regions, indicating important functional roles of noncoding DNA. Understanding the causes of constraint on noncoding sequence evolution forms an important area of research, not least in light of the importance for understanding the evolution of gene expression. We aligned all orthologous genes of chicken and zebra finch together with 5 kb of their upstream and downstream noncoding sequences, to study the evolution of gene flanking sequences in the avian genome. Using ancestral repeats as a neutral reference, we detected significant evolutionary constraint in the 3' flanking region, highest directly after termination (60%) and then gradually decreasing to about 20% 5 kb downstream. Constraint was higher in annotated 3' untranslated regions (UTRs) than in non-UTRs at the same distance from the stop codon and higher in sequences annotated as microRNA (miRNA)-binding sites than in non-miRNA-binding sites within 3' UTRs. Constraint was also higher when estimated for a smaller data set of genes from more closely related songbird species, indicating turnover of functional elements during avian evolution. On the 5' flanking side constraint was readily seen within the first 125 bp immediately upstream of the start codon (34%) and was about 10% for remaining sequence within 5 kb upstream. Analysis of chicken polymorphism data gave further support for the highest constraint directly before and after the translated region. Finally, we found that genes evolving under the highest constraint measured by d(N)/d(S) also had the highest level of constraint in the 3' flanking region. This study broadens the insights into gene flanking sequence evolution by adding new findings from a vertebrate lineage other than mammals.     

A rearranged DNA sequence possibly related to the translocation of immunoglobulin gene segments.

A 5.3 kb EcoRI fragment (T3, abbreviations in ref. 2) has been cloned from DNA of a kappa light chain producing mouse myeloma. The fragment hybridizes to the k' flanking sequences of the J1 gene segment but not to C gene sequences of kappa light chain DNA. Restriction nuclease mapping and partial nucleotide sequencing showed that the fragment consists of sequences from the 5' side of the J1 and form the 3' side of a V gene segment, which apparently had been linked in a genomic rearrangement process. These rearranged flanking sequences are not the flanking sequences of the V and J gene segments which had been joined to form the two kappa light chain genes of the myeloma. Fragments with the hybridization properties of T3 have been found also in two other kappa and one lambda chain producing myelomas. The linking of flanking sequences in the myeloma genome is discussed with respect to the mechanism of recombination between V and J gene segments.     

Structural organization of the human p53 gene. I. Molecular cloning of the human p53 gene

Human p53 gene was cloned from the normal human placenta DNA and DNA from the strain of human kidney carcinoma transplanted into nude mice. Representative gene library from tumor strain of human kidney carcinoma and library of 15 kb EcoRI fragments of DNA from normal human placenta were constructed. Maniatis gene library was also used. Five clones were isolated from kidney carcinoma library; they covered 27 kb and included full-length p53 gene of 19.5 kb and flanking sequences. From normal placenta libraries three overlapped clones were obtained. Restriction map of cloned sequences was constructed and polarity of the p53 gene determined. The first intron of the gene is large (10.4 kb); polymorphic BglII site was observed in this intron, which allows to discriminate between allelic genes. One of these (BglII-) is ten times more abundant that the other (BglII+). Both allelic genes are able to synthesize the 2.8 kb p53 gene.