A new technique for selective identification and mapping of enhancers within long genomic sequences

We report a new experimental method of direct selection, identification, and mapping of potential enhancer sequences within extended stretches of genomic DNA. The method allows simultaneous cloning of a quantity of sequences instead of tedious screening of the separate ones, thus providing a robust and high-throughput approach to the mapping of enhancers. The selection procedure is based on the ability of such sequences to activate a minimal promoter that drives expression of a selective gene. To this end a mixture of short DNA fragments derived from the segment of interest was cloned in a retroviral vector containing the neomycin phosphotransferase II gene under control of a cytomegalovirus (CMV) minimal promoter. The pool of retroviruses obtained was used to infect HeLa cells and then to select neomycin-resistant colonies containing constructs with enhancer-like sequences. The pool of the genomic fragments was rescued by PCR and cloned, forming a library of the potential enhancers. Fifteen enhancer-like fragments were selected from 1-Mb human genome locus, and enhancer activity of 13 of them was verified in a transient transfection reporter gene assay. The sequences selected were found to be predominantly located near 5' regions of genes or within gene introns.     

Interspersion of repetitive and nonrepetitive DNA sequences in the Drosophila melanogaster genome

Cot analysis shows that the haploid Drosophila genome contains 12% rapidly reassociating, highly reiterated DNA, 12% middle repetitive DNA with an average reiteration frequency of 70, and 70% single-copy DNA. The distribution of the middle repetitive sequences in the genome has been studied by an examination in the electron microscope of the structures obtained when middle repetitive sequences present on large DNA strands reassociate and by the hydroxyapatite binding methods developed by Davidson et al. (1973). At least one third by weight of the middle repetitive sequences are interspersed in single-copy sequences. These interspersed middle repetitive sequences have a fairly uniform distribution of lengths from less than 0.5 to 13 kb, with a number average value of 5.6 kb. The average distance between middle repetitive sequences is greater than 13 kb. The data do not exclude the possibility that essentially all of the middle repetitive sequences have the interspersion pattern described above; however, it is possible that some of the middle repetitive sequences of Drosophila are clustered in stretches of length much greater than 13 kb. The interspersion pattern of the middle repetitive sequences in Drosophila is quite different from that which occurs in the sea urchin, in Xenopus, in rat, and probably many other higher eucaryotes.     

Cross-species enhancer prediction using machine learning

Cis-regulatory elements (CREs) are non-coding parts of the genome that play a critical role in gene expression regulation. Enhancers, as an important example of CREs, interact with genes to influence complex traits like disease, heat tolerance and growth rate. Much of what is known about enhancers come from studies of humans and a few model organisms like mouse, with little known about other mammalian species. Previous studies have attempted to identify enhancers in less studied mammals using comparative genomics but with limited success. Recently, Machine Learning (ML) techniques have shown promising results to predict enhancer regions. Here, we investigated the ability of ML methods to identify enhancers in three non-model mammalian species (cattle, pig and dog) using human and mouse enhancer data from VISTA and publicly available ChIP-seq. We tested nine models, using four different representations of the DNA sequences in cross-species prediction using both the VISTA dataset and species-specific ChIP-seq data. We identified between 809,399 and 877,278 enhancer-like regions (ELRs) in the study species (11.6–13.7% of each genome). These predictions were close to the ~8% proportion of ELRs that covered the human genome. We propose that our ML methods have predictive ability for identifying enhancers in non-model mammalian species. We have provided a list of high confidence enhancers at https://github.com/DaviesCentreInformatics/Cross-species-enhancer-prediction and believe these enhancers will be of great use to the community.     

Structure of thymidine kinase gene introduced into mouse Ltk- cells by a new injection method

Pricking, a new injection method developed by Yamamoto et al. (1981), can be used to introduce DNA into cultured cells with high efficiency. Closed circular plasmid DNA containing the cloned HSV-TK gene (pTK-1) was introduced by this method and the structure of DNA in stable transformants was examined. In most clones, the introduced DNA was integrated into the mouse genome in a tandemly repeated form. The possibility of multiple integration via mouse middle repetitive sequences was also examined using the chimeric plasmid with TK genes and middle repetitive sequences (pMRTK-1). Digestion with restriction enzymes showed that the middle repetitive sequence used in this experiment had no effect on the efficiency of transformation, suggesting that this sequence is unable to mediate homologous recombination with mouse genomes.     

Organization of the Euplotes crassus micronuclear genome

Euplotes crassus, like other hypotrichous ciliated protozoa, eliminates most of its micronuclear chromosomal DNA in the process of forming the small linear DNA molecules that comprise the macronuclear genome. By characterizing randomly selected lambda phage clones of E. crassus micronuclear DNA, we have determined the distribution of repetitive and unique sequences and the arrangement of macronuclear genes relative to eliminated DNA. This allows us to compare the E. crassus micronuclear genome organization to that of another distantly related hypotrichous ciliate, Oxytricha nova. The clones from E. crassus segregate into three prevalent classes: those containing primarily eliminated repetitive DNA (Class I); those containing macronuclear genes in addition to repetitive sequences (Class II); and those containing only eliminated unique sequence DNA (Class III). All of the repetitive sequences in these clones belong to the same highly abundant repetitive element family. Our results demonstrate that the sequence organization of the E. crassus and O. nova micronuclear genomes is related in that the macronuclear genes are clustered together in the micronuclear genome and the eliminated unique sequences occur in long stretches that are uninterrupted by repetitive sequences. In both organisms a single repetitive element family comprises the majority of the eliminated interspersed middle repetitive DNA and appears to be preferentially associated with the macronuclear sequence clusters. The similarities in the sequence organization in these two organisms suggest that clustering of macronuclear genes plays a role in the chromosome fragmentation process.     

Localization of the L1Md family of repeated sequences in the mouse albumin-alpha-fetoprotein gene complex.

Studies on the beta-globin gene complex in the mouse have demonstrated the existence of repeated DNA sequences interspersed throughout the intergenic regions (1,2). These sequences are members of families of middle repetitive sequences and have been mapped to specific intergenic sites in the 60 kbp beta-globin complex. In this study we present evidence that members of this middle repetitive family of DNA sequences, the L1Md family, are interspersed throughout the mouse albumin and alpha-fetoprotein gene complex. Unlike those of the beta-globin complex, all of which are found in the intergenic regions, these sequences are localized within intron 12 of the albumin gene and intron 3 of the AFP gene as well as twice in the 13.5 kbp intergenic region that links the albumin gene to the AFP gene.     

DNA synthesized at pachytene in Lilium: A non-divergent subclass of moderately repetitive sequences

Moderately repetitive sequences of Lilium DNA synthesized during pachytene consist of families that have a considerably lower divergence than those of total genomic middle repeat DNA, the latter having an average divergence of 10%. 80% of the sequences synthesized during the early phase of pachytene and 100% of those synthesized during the latter phase of pachytene reassociate with perfect or near-perfect fidelity. Except for the small amount of DNA synthesized during early pachytene, pachytene middle repeat sequences are non-divergent and thus constitute a distinctive subset of total moderately repetitive DNA. The modal length of pachytene and total middle repeat sequences are similar. In contrast to earlier measurements based on isotope incorporation, the modal length is of the order of 1500–2000 base pairs, ten times the previously estimated size. Calculations based on the new length lead to the conclusion that pachytene middle repeat sequences account for 1% of the genome.     

Isolation and structural analysis of ribosomal protein genes in Xenopus laevis. Homology between sequences present in the gene and in several different messenger RNAs

The ribosomal protein genes are present in two to four copies per haploid genome of Xenopus laevis. Using cloned complementary DNA probes, we have isolated, from a genomic library of X. laevis, several clones containing genes for two different ribosomal proteins (L1 and L14). These genes contain intervening sequences. In the case of the L1 gene, the exons are 100 to 200 base-pairs long and the introns, on average, 400 base-pairs. Along the genomic fragments, two different classes of repetitive DNA are present: highly and middle repetitive DNA. Both are evolutionarily unstable as shown by hybridization to Xenopus tropicalis DNA. Several introns of the gene coding for protein L1 contain middle repetitive sequences. Hybridization and hybrid-released translation experiments have shown that sequences inside the two genes hybridize to several poly(A) messenger RNAs. Some of the products encoded by these mRNA have electrophoretic properties of ribosomal proteins.     

Sex-specific organisation of middle repetitive DNA sequences in the mealybug Planococcus lilacinus.

Differential organisation of homologous chromosomes is related to both sex determination and genomic imprinting in coccid insects, the mealybugs. We report here the identification of two middle repetitive sequences that are differentially organised between the two sexes and also within the same diploid nucleus. These two sequences form a part of the male-specific nuclease-resistant chromatin (NRC) fraction of a mealybug Planococcus lilacinus. To understand the phenomenon of differential organisation we have analysed the components of NRC by cloning the DNA sequences present, deciphering their primary sequence, nucleosomal organisation, genomic distri-bution and cytological localisation. Our observations suggest that the middle repetitive sequences within NRC are functionally significant and we discuss their probable involvement in male-specific chromatin organisation.     

Organization of the Euplotes crassus Micronuclear Genome1

Euplotes crassus, like other hypotrichous ciliated protozoa, eliminates most of its micronuclear chromosomal DNA in the process of forming the small linear DNA molecules that comprise the macronuclear genome. By characterizing randomly selected lambda phage clones of E. crassus micronuclear DNA, we have determined the distribution of repetitive and unique sequences and the arrangement of macronuclear genes relative to eliminated DNA. This allows us to compare the E. crassus micronuclear genome organization to that of another distantly related hypotrichous ciliate, Oxytricha nova. The clones from E. crassus segregate into three prevalent classes: those containing primarily eliminated repetitive DNA (Class I); those containing macronuclear genes in addition to repetitive sequences (Class II); and those containing only eliminated unique sequence DNA (Class III). All of the repetitive sequences in these clones belong to the same highly abundant repetitive element family. Our results demonstrate that the sequence organization of the E. crassus and O. nova micronuclear genomes is related in that the macronuclear genes are clustered together in the micronuclear genome and the eliminated unique sequences occur in long stretches that are uninterrupted by repetitive sequences. In both organisms a single repetitive element family comprises the majority of the eliminated interspersed middle repetitive DNA and appears to be preferentially associated with the macronuclear sequence clusters. The similarities in the sequence organization in these two organisms suggest that clustering of macronuclear genes plays a role in the chromosome fragmentation process.     

The organization of DNA sequences in the mouse genome

Analysis of the organization of nucleotide sequences in mouse genome is carried out on total DNA at different fragment size, reannealed to intermediate value of Cot, by Ag⁺-Cs₂SO₄ density gradient centrifugation. — According to nuclease S-1 resistance and kinetic renaturation curves mouse genome appears to be made up of non-repetitive DNA (76% of total DNA), middle repetitive DNA (average repetition frequency 2×10⁴ copies, 15% of total DNA), highly repetitive DNA (8% of total DNA) and fold-back DNA (renatured density 1.701 g/ml, 1% of total DNA).— Non-repetitive sequences are intercalated with short middle repetitive sequences. One third of non-repetitive sequences is longer than 4500 nucleotides, another third is long between 1800 and 4500 nucleotides, and the remainder is shorter than 1800 nucleotides. —Middle repetitive sequences are transcribed in vivo. The majority of the transcribed repeated sequences appears to be not linked to the bulk of non-repeated sequences at a DNA size of 1800 nucleotides. — The organization of mouse genome analyzed by Ag⁺-Cs₂SO₄ density gradient of reannealed DNA appears to be substantially different than that previously observed in human genome using the same technique.     

DNA sequence repetition in the genome of the American oyster.

The genome of Crassostrea virignica, the American oyster, has been studied by reassociation kinetics in order to construct a profile of DNA sequence frequency components. Oyster DNA has been shown to contain at least 51% single copy DNA sequences and two classes of middle repetitive DNA. The major repetitive class contains sequences which are repeated on the average 20 times and comprise 29% of oyster DNA. The other class represents 10% of oyster DNA and contains sequences repeated approx. 3000 times. In addition the DNA of oyster contains at least 1% foldback sequences. The spectrum of DNA repetition components in the American oyster is similar to that found in the genomes of other mollusks.     

Characterization of the nuclear genome of pearl millet.

The nuclear genome of pearl millet has been characterized with respect to its size, buoyant density in CsCl equilibrium density gradients, melting temperature, reassociation kinetics and sequence organization. The genome size is 0.22 pg. The mol percent G + C of the DNA is calculated from the buoyant density and the melting temperature to be 44.9 and 49.7%, respectively. The reassociation kinetics of fragments of DNA 300 nucleotides long reveals three components: a rapidly renaturing fraction composed of highly repeated and/or foldback DNA, middle repetitive DNA and single copy DNA. The single copy DNA consists of 17% of the genome. 80% of the repetitive sequences are at least 5000 nucleotide pairs in length. Thermal denaturation profiles of the repetitive DNA sequences show high Tm values implying a high degree of sequence homogeneity. About half of the single copy DNA is short (750--1400 nucleotide paris) and interspersed with long repetitive DNA sequences. The remainder of the single copy sequences vary in size from 1400 to 8600 nucleotide pairs.     

Chromatin domain boundary element search tool for Drosophila

Chromatin domain boundary elements prevent inappropriate interaction between distant or closely spaced regulatory elements and restrict enhancers and silencers to correct target promoters. In spite of having such a general role and expected frequent occurrence genome wide, there is no DNA sequence analysis based tool to identify boundary elements. Here, we report chromatin domain Boundary Element Search Tool (cdBEST), to identify boundary elements. cdBEST uses known recognition sequences of boundary interacting proteins and looks for 'motif clusters'. Using cdBEST, we identified boundary sequences across 12 Drosophila species. Of the 4576 boundary sequences identified in Drosophila melanogaster genome, >170 sequences are repetitive in nature and have sequence homology to transposable elements. Analysis of such sequences across 12 Drosophila genomes showed that the occurrence of repetitive sequences in the context of boundaries is a common feature of drosophilids. We use a variety of genome organization criteria and also experimental test on a subset of the cdBEST boundaries in an enhancer-blocking assay and show that 80% of them indeed function as boundaries in vivo. These observations highlight the role of cdBEST in better understanding of chromatin domain boundaries in Drosophila and setting the stage for comparative analysis of boundaries across closely related species.     

Distribution pattern and enzymic hypermethylation of inverted repetitive DNA sequences in P815 mastocytoma cells.

A specific class of DNA sequences, the inverted repetitive sequences, forms a double-stranded structure within a single linear polynucleotide chain in denatured DNA. The reassociation process is unimolecular and occurs very fast. Quantitative analyses have shown that in mouse P815 cells these sequences comprise about 4% of the nuclear DNA and are interspersed within sequences of other degrees of repetitiveness. After labeling the cells with L-[Me-3H]methionine and [14C]deoxycytidine, relative rates of enzymic DNA methylation were computed on the basis of radioactivities found in pyrimidine residues of the nuclear DNA. The results indicate that in P815 cells, DNA of inverted repetitive sequences is methylated to a level about 50% higher than the normal repetitive DNA sequences and to about 300% higher than the unique and intermediary intermediatry sequences. The biological function of the inverted repetitive sequences, as well as of the role of enzymic methylation of DNA remains unknown.