The amino acid sequence of Lrp is highly conserved in four enteric microorganisms.

Lrp (leucine-responsive regulatory protein) is a global regulator of metabolism in Escherichia coli (J. M. Calvo and R. G. Matthews, Microbiol. Rev. 58:466-490, 1994). The lrp genes from three other enteric microorganisms, Enterobacter aerogenes, Klebsiella aerogenes, and Salmonella typhimurium, were cloned and sequenced. An analysis of these sequences and of the previously determined sequence from E. coli indicated that the vast majority of changes were synonymous rather than nonsynonymous changes. Nucleotide changes occurred at 89 of 492 positions but resulted in amino acid changes at only 2 of 164 positions. This analysis suggests that the Lrp amino acid sequence is highly adapted for function and that almost all amino acid changes lead to a protein that functions less well than the wild-type protein.     

DSCAM, a highly conserved gene in mammals, expressed in differentiating mouse brain

Down Syndrome Cell Adhesion molecule (DSCAM) is a member of the immunoglobulin superfamily, and represents a novel class of neuronal cell adhesion molecules. In order to understand the cellular functions of DSCAM, we isolated full-length mouse and human cDNA clones, and analysed its expression during mouse development and differentiation. Sequence analysis of the human DSCAM cDNA predicted at least 33 exons that are distributed over 840 kb. When compared to human DSCAM, the mouse homologue showed 90 and 98% identity at the nucleotide and amino acid levels, respectively. In mouse, DSCAM is located on 16C, the syntenic region for human chromosome band 21q22 and also the region duplicated in mouse DS models. DSCAM gene is predicted to encode an approximately 220-kDa protein, and its expression shows dynamic changes that correlate with neuronal differentiation during mouse development. Our results suggest that DSCAM may play critical roles in the formation and maintenance of specific neuronal networks in brain.     

The 1723 element: a long, homogeneous, highly repeated DNA unit interspersed in the genome of Xenopus laevis

We describe a highly repeated DNA element in the Xenopus laevis genome. This sequence, named the 1723 element, was first identified among sequences that are transcribed during embryonic development. The element is present in about 8500 copies per haploid genome, which together accounts for about 2.4% of the genome. Most copies of the element have highly conserved restriction maps, and are interspersed in the genome. The copies range in size from 6000 to 10,000 base-pairs due to an expandable region that contains variable numbers of a tandemly repeating 183 to 204 base-pair unit. The element is framed by an imperfect 18 base-pair inverted sequence, and inverted repeats of 180 to 185 base-pairs are nearby. Sequence analysis of DNA adjacent to three cloned elements shows that the elements are flanked by 8 base-pair direct repeats. These and other properties of 1723 suggest that it may be transposable.     

Distribution of repetitious DNA in randomly growing and synchronized Chinese hamster cells.

Early spermatogenic cells from the testes of 10-, 13-, 15-, 18-, 20- and 25-day-old rats were purified by sedimentation at unit gravity. Cell dissociation was accomplished in 5 mM EDTA or 0.1% trypsin in Ca-Mg-free phosphate-buffered saline (pH 7.35). Dissociation with trypsin resulted in more viable cells than with EDTA, while EDTA was more efficient for the dissociation of spermatogonia. The differential effects of the two dissociation media were particularly evident in cell preparations from the 10-day-old animal. Maximum purity of different cell types was obtained in different aged animals (spermatogonia, 98%, 10 days; preleptotene spermatocytes, 98%, 10 days; leptotene spermatocytes, 75%, 13 days; zygotene spermatocytes, 68%, 18 days; pachytene spermatocytes, 75%, 25 days). Purity of particular types was correlated with the age of the animal. Earlier stages were purified to a greater extent in younger animals and later stages to a greater extent in older animals. Later stages exhibited increasing sedimentation at unit gravity in correlation with the increase in cell size as differentiation proceeded to pachytene spermatocyte. Two early germinal cell types, spermatogonia and preleptotene spermatocytes, were greatly purified with this technique.     

The YYRR box: a conserved dipyrimidine-dipurine sequence element in Drosophila and other eukaryotes.

We have discovered a novel DNA sequence element in Drosophila which is based upon a CTGA tandem repeat. This element has been named the YYRR box to emphasize its dipyrimidine-dipurine nature which is predicted to have unusual structural features. Southern hybridization analysis of genomic DNA indicates the presence of 25-30 copies of the YYRR box in each of three Drosophila species (melanogaster, pseudoobscura, and virilis) and conservation of genomic location within species. Similar analysis of human and rat DNA indicates the presence of YYRR related sequences in mammals as well. YYRR boxes have been localized to two genetic loci in Drosophila: Gld and a gene tentative identified as ted. These two genes exhibit correlated patterns of developmental expression and an identical mutant phenotype. Sequence analysis of the Gld YYRR box in three Drosophila species revealed a high degree of conservation despite its intronic location.     

A host cell protein binds to a highly conserved sequence element (pac-2) within the cytomegalovirus a sequence.

The human cytomegalovirus (CMV) a sequence has significant homology to two regions, pac-1 and pac-2, within the a sequence of herpes simplex virus type 1 (HSV-1). Both regions have been shown to be important cis-acting signals in HSV-1 genome maturation. We have demonstrated that a small fragment from within the CMV a sequence, containing the pac-1 and pac-2 motifs, carries all of the signals necessary for generation of genomic termini and for inversion. These observations indicated that the function of these highly conserved sequence motifs was similar in CMV and HSV-1. We have identified and partially purified a host cell protein with affinity for the sequence 5'-GGCGGCGGCGCATAAAA-3' within CMV pac-2. This partially purified protein has an apparent molecular weight of 89,000 under denaturing conditions and could be renatured after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the capacity to bind DNA was the property of a single polypeptide chain. This activity was found in a wide variety of human cell lines, including those that are permissive as well as those that are nonpermissive for CMV growth, but not in cell lines from monkey, mouse, or drosophila origins. Our work implicates a host cell protein in a sequence function.     

The family of highly interrelated single-stranded deoxyribonucleic acid plasmids.

Many plasmids from gram-positive bacteria replicate via a single-stranded deoxyribonucleic acid (ssDNA) intermediate, most probably by a rolling-circle mechanism (these plasmids are referred to in this paper as ssDNA plasmids). Their plus and minus origins are physically separated, and replicative initiations are not simultaneous; it is this feature that allows visualization of ssDNA replication intermediates. The insertion of foreign DNA into an ssDNA plasmid may provoke a high frequency of deletions, changes of replicative products to high-molecular-weight forms, segregational loss, and decreased plasmid copy numbers. When an ssDNA plasmid is inserted into the chromosome, both deletions and amplifications may be induced. Both the mode of replication and the copy control mechanism affect the fate of inserted foreign material, usually selecting for its loss. Thus, after having tasted various morsels of DNA, the resulting plasmid stays trim. The features of the ssDNA plasmids seem to be beneficial for their viability and propagation, but not for their use as cloning vectors. However, plasmids replicating via ssDNA intermediates are being exploited to yield insights into the mechanisms of recombination and amplification.     

The ORFa protein, the putative transposase of maize transposable element Ac, has a basic DNA binding domain.

Ac encodes the 807 amino acid ORFa protein which binds specifically to multiple AAACGG motifs that are subterminally located in both ends of Ac. The wild-type ORFa protein and a number of deletion and amino acid exchange mutants were expressed in Escherichia coli, renatured and used for mobility shift assays. At least 136 amino acids from the N-terminus and 537 C-terminal amino acids may be removed from the ORFa protein without destroying the DNA binding domain, whereas a protein starting at amino acid 189 is DNA binding deficient. Certain basic amino acids between positions 190 and 200 are essential for DNA binding, as their substitution with uncharged amino acids leads to the loss of this function. The DNA binding domain of ORFa protein has an overall basic character, but no obvious sequence homology to any other known DNA binding protein. The homologies to the major open reading frames of transposable elements Tam3 from Antirrhinum majus and Hobo from Drosophila are found between the C-terminal two thirds of the three proteins. The ORFa protein forms discrete complexes with target DNA that appear, depending on the protein concentration, as a 'ladder' of bands on gels, indicating the occupation of target DNA by multiple ORFa protein molecules.     

A germline restricted, highly repetitive DNA sequence in Paramyxineatami: an interspecifically conserved, but somatically eliminated, element

In some species of hagfish, the phenomenon of chromosome elimination occurs during embryogenesis. However, only two repetitive DNA families are known to be represented in chromosomes that are eliminated from somatic cells of the Japanese hagfish Eptatretus okinoseanus. Using molecular analyses, another germ line-restricted, highly repetitive DNA family has been detected in another Japanese hagfish, Paramyxine atami. The repeat unit of this family, which is 83?bp long, has been designated “EEPa1”, for Eliminated Element of P. atami 1. DNA filter hybridization using EEPa1 as a probe revealed that this family is shared among several species and is conserved in the germline DNA. Although eliminated, repetitive DNA that is shared interspecifically has not been reported in hagfish species, cases of chromatin diminution and chromosome elimination processes have been described previously in other organisms.The patterns and intensities of hybridization signals suggest that members of the repetitive DNA family defined by EEPa1 have undergone concerted molecular evolution.     

Evolutionarily conserved structure of the 3' non-translated region of a Chinese hamster polyubiquitin gene.

From a V79 Chinese hamster genomic library, we isolated a clone containing a polyubiquitin gene (designated as CHUB1), and determined its nucleotide sequence. The coding region of the CHUB1 gene consisted of five direct repeats of the ubiquitin unit with no spacer, followed by a single tyrosine residue. Northern hybridization analysis with a synthesized probe specific to the 3' non-translated region of the CHUB1 gene revealed that it codes for a 1.8 kb mRNA. An evident homology to the human polyubiquitin gene UbB and the chicken UbI gene was observed in the region corresponding to the full extent of the mature mRNA sequence, suggesting that these three genes belong to a common polyubiquitin gene subfamily, and that the sequence in the 3' non-translated region of the CHUB1 gene is unique to this subfamily.     

The effects of deoxyribonucleic acid secondary structure on tertiary structure.

The secondary structure of supercoiled DNA was varied by changes in ionic strength. For I = 0.075-0.4 the structure remained in the previously established branched form with only minor alterations in molecular dimensions. In 4M-NaCl, which induces linear DNA to change its secondary structure to the C structure and brings about an increase in the superhelix density of the molecule, no extra branches were observed on the molecules. The limiting factors that dictate supercoil structure seem to be the number and position of potential branch points and the proximity with which the two intertwining DNA strands can approach each other on the arms of the branches. This value is close to 10nm under the conditions described, and is 14-15nm at I = 0.2. It is suggested that such values should be borne in mind when models of chromosome structure are being constructed.     

The sequence, structure and evolutionary features of HOTAIR in mammals

Background  

An increasing number of long noncoding RNAs (lncRNAs) have been identified recently. Different from all the others that function in cis to regulate local gene expression, the newly identified HOTAIR is located between HoxC11 and HoxC12 in the human genome and regulates HoxD expression in multiple tissues. Like the well-characterised lncRNA Xist, HOTAIR binds to polycomb proteins to methylate histones at multiple HoxD loci, but unlike Xist, many details of its structure and function, as well as the trans regulation, remain unclear. Moreover, HOTAIR is involved in the aberrant regulation of gene expression in cancer.     

Recognition sequence of a highly conserved DNA binding protein RBP-J kappa.

DNA binding specificity of the RBP-J kappa protein was extensively examined. The mouse RBP-J kappa protein was originally isolated as a nuclear protein binding to the J kappa type V(D)J recombination signal sequence which consisted of the conserved heptamer (CACTGTG) and nonamer (GGTTTTTGT) sequences separated by a 23-base pair spacer. Electrophoretic mobility shift assay using DNA probes with mutations in various parts of the J kappa recombination signal sequence showed that the RBP-J kappa protein recognized the sequence outside the recombination signal in addition to the heptamer but did not recognize the nonamer sequence and the spacer length at all. Database search identified the best naturally occurring binding motif (CACTGTGGGAACGG) for the RBP-J kappa protein in the promoter region of the m8 gene in the Enhancer of split gene cluster of Drosophila. The binding assay with a series of m8 motif mutants indicated that the protein recognized mostly the GTGGGAA sequence and also interacted weakly with ACT and CG sequences flanking this hepta-nucleotide. Oligonucleotides binding to the RBP-J kappa protein were enriched from a pool of synthetic oligonucleotides containing 20-base random sequences by the repeated electrophoretic mobility shift assay. The enriched oligomer shared a common sequence of CGTGGGAA. All these data indicate that the RBP-J kappa protein recognizes a unique core sequence of CGTGGGAA and does not bind to the V(D)J recombination signal without the flanking sequence.