Banded krait minor-satellite (Bkm)-associated Y chromosome-specific repetitive DNA in mouse.

The mouse Y chromosome remains highly condensed in all somatic tissues but decondenses extensively in testis. We have isolated a mouse Y chromosome-specific repeat M34 (11.5 kb) and shown that this is distributed along the Y chromosome except the sex-determining region (the Y short arm) in which GATA repeats are predominantly concentrated. It has 32 copies of GATA repeats in a 2.7 kb fragment. About 200-300 copies of M34 on the Y chromosome are interspersed among other sequences. A 1.2 kb fragment (p3) of M34, containing GATA repeats, also has scaffold attachment region (SAR) motifs which bind to nuclear matrices. A strong affinity of histone H1 to SAR motifs is implicated in maintaining the condensed state of the Y chromosome in somatic tissues. The probable significance of molecular organization of the Y chromosome is discussed.     

Sense in antisense?

A correspondence between open reading frames in sense and antisense strands is expected from the hypothesis that the prototypic triplet code was of general form RNY, where R is a purine base, N is any base, and Y is a pyrimidine. A deficit of stop codons in the antisense strand (and thus long open reading frames) is predicted for organisms with high G + C percentages; however, two bacteria (Azotobacter vinelandii, Rhodobacter capsulatum) have larger average antisense strand open reading frames than predicted from (G + C)%. The similar Codon frequencies found in sense and antisense strands can be attributed to the wide distribution of inverted repeats (stem-loop potential) in natural DNA sequences.     

Molecular cloning and characterization of a complete Chinese hamster provirus related to intracisternal A particle genomes.

We report here the nucleotide sequence of a full-length Chinese hamster genomic proviral element, CHIAP34. CHIAP34 is 6,403 bp long with long terminal repeats of 311 bp at each end. The genetic organization of CHIAP34 was determined by comparison with intracisternal A particle (IAP) genetic elements from the mouse and Syrian hamster. Extensive homology at the nucleotide and deduced amino acid sequence levels was observed between CHIAP34 and the mouse and Syrian hamster IAP elements. CHIAP34 may represent a defective Chinese hamster IAP genetic element. The gag gene consists of 837 codons, of which 558 codons are in a single long open reading frame followed by several frameshifts. The pol gene begins with a -1 frameshift and consists of a long open reading frame of 753 codons followed by a short open reading frame of 103 codons. The putative env region contains multiple termination codons in all reading frames. CHIAP34 is representative of the predominant retroviral elements in the Chinese hamster ovary cell genome present at around 80 copies per haploid genome.     

Nucleotide sequence of a complete mouse intracisternal A-particle genome: relationship to known aspects of particle assembly and function.

The 7,095-nucleotide sequence of a mouse genomic intracisternal A-particle (IAP) element, MIA14, is reported. MIA14 is known to be colinear with IAP 35S RNA and to contain functional long terminal repeats. Its internal genetic organization was determined by comparisons with a homologous Syrian hamster element and the related retroviruses simian retrovirus 1 (simian type D) and Rous sarcoma virus (avian type C). MIA14 contains a gag-protease open reading frame of 827 codons and a pol region of 867 codons entered by a frame shift of -1. The env region of 1,100 base pairs has multiple stop codons in all reading frames, consistent with the failure thus far to detect IAP-related glycosylated envelope components. RNA transcribed in vitro from a cDNA clone containing a closely homologous gag-protease open reading frame was translated in a cell-free system. The main product was a 73-kilodalton polypeptide immunoprecipitable with antiserum against the authentic IAP gag-related structural protein p73. Rather than ending at the gag-protease boundary, p73 appears to contain 7 to 8 kilodaltons of peptide encoded by the protease domain, a peculiarity possibly related to the observed impairment of normal protein processing in IAPs. The N-terminal 217 codons of gag are unique to murine IAPs and may have been contributed by recombination with a cellular gene. The mouse-specific region of gag encodes a hydrophobic signal peptide with an atypical cleavage site. Delayed cleavage of this peptide could result in anchoring of newly synthesized p73 to the endoplasmic reticulum membrane and restriction of particle assembly to this site.     

A frame-specific symmetry of complementary strands of DNA suggests the existence of genes on the antisense strand

The bacterial DNA sequence in GenBank database were divided into coding and noncoding regions and examined for the base-trimer distribution in every triplet frame on the sense and antisense strands. The results revealed that for the noncoding region, both strands have very similar base-trimer distributions and have no frame specificity; that is, DNA is symmetric in the noncoding region. For the coding region, on the other hand, the symmetry is broken only in the triplet framework, and we found a special triplet-frame-specific symmetry which appears when the two complementary strands of the coding region are read from their 5 ends. In addition, the following frame specificity was also observed in the distribution of stop codons on the antisense strand of the coding region. When the antisense sequences of the open reading frames (ORFs) in the database are read in the three reading frames, the same reading frame as the corresponding ORF contains a significantly larger amount of long open frames without stop codons (i.e., nonstop frames [NSFs]) than expected, while the number of NSFs in the other two reading frames is similar to that of the expected one. That is, NSFs as well as ORFs are maintained in a frame-specific manner, and in this sense, DNA becomes symmetrical even in the coding region. These two kinds of frame-specific symmetries indicate that only an ORF and its complementary triplets are specifically recognized and maintained in DNA. We suppose that the antisense strands as well as the sense strands in the coding region may be transcribed, thereby producing various kinds of proteins corresponding to NSFs, though their amount may not be large. The presence of these proteins should have some benefits for living organisms, and therefore we propose that these proteins are upcoming enzymes having novel functions.Correspondence to: I. Urabe     

Nucleotide sequence of cauliflower mosaic virus DNA

The complete nucleotide sequence (8024 nucleotides) of the circular double-stranded DNA of cauli-flower mosaic virus has been established. The DNA molecule is known to possess three discrete single-stranded discontinuities, often referred to as “gaps”, two in one strand and one in the other. The sequence data indicate that gap 1, the single discontinuity in the α strand, corresponds to the absence of no more than one or two nucleotides with respect to the complementary β strand. The two discontinuities in the β strand, however, are not authentic gaps since no nucleotides are missing, but are instead regions of sequence overlap: a short sequence (19 residues for gap 2, at least 2 residues for gap 3) at one terminus of each discontinuity, probably the 5′ terminus, is displaced from the double helix by an identical sequence at the other boundary of the discontinuity. Analysis of the distribution of nonsense codons in the DNA sequence is consistent with other evidence that only the α strand is transcribed. The coding region extends around the circular molecule from 4 map units of gap 1, the map origin, to map position 91, and consists of six long open reading frames. Our findings suggest, but do not prove, that the DNA sequence of the open reading frames is colinear with viral protein sequences. The cistron for the viral coat protein, which is probably synthesized in the form of a precursor, has been situated in coding region IV on the basis of its unusual amino acid composition.     

Identification of the functional initiation codons of a phase-variable gene of Haemophilus influenzae, lic2A, with the potential for differential expression

Simple sequence repeats located within reading frames mediate phase-variable ON/OFF switches in gene expression by generating frameshifts. Multiple translation initiation codons in different reading frames are found upstream of most Haemophilus influenzae tetranucleotide repeat tracts, raising the possibility of multiple active reading frames and more than two levels of gene expression for these loci. Phase variation between three levels of gene expression (strong, weak, and none) was observed when lic2A was fused to a lacZ reporter gene. The lic2A 5' CAAT repeat tract is preceded by four 5' ATG codons (x, y, z1, and z2) in two reading frames. Each of these initiation codons was inactivated by site-directed mutagenesis. Strong expression from frame 1 was associated with x but not y. Weak expression from frame 2 was mainly dependent on the z2 codon, and there was no expression from frame 3. Using monoclonal antibodies specific for a digalactoside epitope of lipopolysaccharide whose synthesis requires Lic2A, two levels (strong and undetectable) of antibody reactivity were detected, suggesting that weak expression of lic2A is not discernible at the phenotypic level. Inactivation of the x initiation codon resulted in loss of strong expression of the digalactoside epitope and elevated killing by human serum. The failure to detect more than two phenotypes for lic2A, despite clear evidence of weak expression from the z1/z2 initiation codons, leaves open the question of whether or not multiple initiation codons are associated with more complex patterns of phenotypic variation rather than classical phase-variable switching between two phenotypes.     

A model for microtubule-associated protein 4 structure. Domains defined by comparisons of human, mouse, and bovine sequences.

cDNAs encoding human and mouse microtubule-associated protein 4 (MAP 4) were isolated. MAP 4 is encoded by a single gene. Multiple MAP 4 mRNAs are transcribed that are differentially expressed among mouse tissues. Open reading frames for the human and mouse MAP 4 clones indicate three distinct regions consisting of related sequences with different motifs. Approximately 30% of the protein is tandem related repeats of approximately 14 amino acids. Another region contains clusters of serine and proline. Four 18-mer repeats characteristic of the microtubule-binding domains of MAP 2 and tau are located at the carboxyl-terminal portion of MAP 4. Amino acid sequence analysis revealed that human and mouse MAP 4 are homologs of the bovine 190-kDa MAP/MAP U (Aizawa, H., Emori, Y., Murofushi, H., Kawasakai, H., Sakai, H., and Suzuki, K. (1990) J. Biol. Chem. 265, 13849-13855). Mouse and human MAP 4 and the bovine 190-kDa MAP are approximately 75% similar, indicating that these proteins are all members of the same class. Domains with extremely high conservation (greater than or equal to 88%) are: 1) the extreme amino terminus; 2) a proline-rich region between the KDM and S,P domains; 3) the microtubule-binding domain; and 4) the extreme carboxyl terminus.     

Comparison of the maxicircle (mitochondrial) genomes of Leishmania tarentolae and Trypanosoma brucei at the level of nucleotide sequence

The entire 16.7-kilobase (kb) transcribed region of the Leishmania tarentolae maxicircle was compared to the entire 15-kb transcribed region of the Trypanosoma brucei maxicircle at the nucleotide sequence level by dot matrix analysis and by alignments of individual genes. The L. tarentolae NADH dehydrogenase subunit 1 (ND1) gene was identified in a newly obtained 2.9-kb sequence. All but two regions which flank the cytochrome b gene are highly conserved in both species. One 3.1-kb region in L. tarentolae that contains the cytochrome oxidase subunit III (COIII) gene and several open reading frames corresponds to a 2-kb sequence in T. brucei with limited sequence homology that lacks the COIII gene. Another 0.6-kb region that comprises an unidentified open reading frame (open reading frame 12) in L. tarentolae is substituted by a nonhomologous 0.4-kb open reading frame in T. brucei. A short intergenic region between the ND1 gene and the maxicircle unidentified reading frame 1 gene shows limited sequence homology, and the regions between the ND4 and ND5 genes and between the COI and ND4 genes are not conserved. All of the intergenic regions share G + C richness and a similar pattern of G versus C strand bias. 1.8 kb of the L. tarentolae divergent region (DV) and around 3 kb of the T. brucei DV were also obtained. The T. brucei DV sequences were not homologous to the L. tarentolae DV sequence but were organized in a similar fashion with tandem repeats of varying complexity.     

Receptor-like genes in the major resistance locus of lettuce are subject to divergent selection.

Disease resistance genes in plants are often found in complex multigene families. The largest known cluster of disease resistance specificities in lettuce contains the RGC2 family of genes. We compared the sequences of nine full-length genomic copies of RGC2 representing the diversity in the cluster to determine the structure of genes within this family and to examine the evolution of its members. The transcribed regions range from at least 7.0 to 13.1 kb, and the cDNAs contain deduced open reading frames of approximately 5. 5 kb. The predicted RGC2 proteins contain a nucleotide binding site and irregular leucine-rich repeats (LRRs) that are characteristic of resistance genes cloned from other species. Unique features of the RGC2 gene products include a bipartite LRR region with >40 repeats. At least eight members of this family are transcribed. The level of sequence diversity between family members varied in different regions of the gene. The ratio of nonsynonymous (Ka) to synonymous (Ks) nucleotide substitutions was lowest in the region encoding the nucleotide binding site, which is the presumed effector domain of the protein. The LRR-encoding region showed an alternating pattern of conservation and hypervariability. This alternating pattern of variation was also found in all comparisons within families of resistance genes cloned from other species. The Ka /Ks ratios indicate that diversifying selection has resulted in increased variation at these codons. The patterns of variation support the predicted structure of LRR regions with solvent-exposed hypervariable residues that are potentially involved in binding pathogen-derived ligands.     

Upstream open reading frames regulate the translation of the multiple mRNA variants of the estrogen receptor alpha

It is by now well established that the estrogen receptor alpha (ER alpha) is transcribed from multiple promoters. One direct consequence of multiple promoters is the generation of mRNA variants with different 5'-untranslated regions (5'-UTRs). However, the potential roles of these individual mRNA variants are not known. All 5'-UTRs of ER alpha contain between one and six upstream open reading frames. In this study the effect of the 5'-UTRs of major human and mouse ER alpha mRNA variants on translation was evaluated. Some of the 5'-UTRs were found to strongly inhibit translation of the downstream open reading frame. Mutation of the upstream AUG codons partially or completely restored translation efficiency. A toeprinting analysis and assessment of the contribution of each AUG codon to the inhibitory effect on translation showed that leaky scanning and reinitiation occurs with these mRNAs. In conclusion, the upstream open reading frames in the 5'-UTRs of ER alpha mRNAs have the potential to regulate estrogen receptor alpha expression.     

Statistical evaluation of the coding capacity of complementary DNA strands

Two independent methods are used to evaluate the protein-coding information content in different classes of DNA sequences. The first method allows to evaluate the statistical relevance of finding unidentified reading frames, longer than 100 codons, on both DNA strands of: a) 117 DNA sequences that code for 142 nuclear proteins; b) 39 stable RNA coding sequences and c) 36 other DNA sequences which include regulatory and as yet unknown function sequences. The finding of 50 reading frames longer than 100 codons (complementary inverted proteins or c.i.p. genes) located on the DNA strand complementary to the protein-coding one is drastically in excess of the number predicted by chance alone. An independent method (testcode) applied to c.i.p. gene sequences, which assigns the probability of coding to a given sequence, predicts that more than 50% of these genes are translated in a functional product. These analyses indicate the existence of a new class of protein-coding genes, located on the DNA sequences complementary to the protein-coding DNA strand.     

Folbos, a new foldback element in rice

A new class I foldback element, Folbos, has been discovered in O. sativa L. Its long terminal inverted repeats (IVRs) are 303 and 331 bp long and the left one encodes a short open reading frame of 76 codons. The IVRs consist of inner and outer domains, the latter built up of 6 tandem repeats of about 30 bp each. The central region is represented by 90 bp conservative stretch adjacent to a variable length (19-33 bp) A-tail, which in most cases includes the sequence 5'-TGACTT-3'. Folbos targets AT-rich regions and the insertion results in 7 bp target site duplications. Half of the copies found in annotated sequences of O. sativa japonica cv. Nipponbare are positioned in close proximity to (< 1kb) or within the transcribed regions, thus they have the potential to contribute to plant genome evolution.