Human papillomavirus 1a complete DNA sequence: a novel type of genome organization among Papovaviridae

[This corrects the article on p. 231 in vol. 1, PMID: 6325156.].     

Human papillomavirus 1a complete DNA sequence: a novel type of genome organization among papovaviridae

The complete nucleotide sequence of human papillomavirus type 1a (7811 nucleotides) has been established. The overall organization of the viral genome is different from that of other related papovaviruses (SV40, BKV, polyoma). Firstly, genetic information seems to be coded by one strand. Secondly, no significant homology is found with SV40 or polyoma coding sequence for either DNA or deducted protein sequences. The relatedness of human and bovine papillomaviruses is revealed by a conserved coding sequence in the two species. Two regions can be defined on the viral genome: the putative early region contains two large open reading frames of 1446 and 966 nucleotides, together with several split ones, and corresponds to the transforming part of the bovine papillomavirus type 1 genome, and the remaining sequences, which include two open reading frames likely to encode structural polypeptide(s). The DNA sequence is analysed and putative signals for regulation of gene expression, and homologies with the Alu family of human ubiquitous repeats and the SV40 72-bp repeat are outlines.     

Isolation and characterization of human papillomavirus type 6-specific T cells infiltrating genital warts.

The potential role of T cells in the control of human papillomavirus type 6 (HPV-6) infections is an appealing premise, but their actual role has been sparsely investigated. Since HPV-6 infections are confined to the epithelium, such an investigation should focus on the T cells present at the site of infection (i.e., the warts). Therefore, we isolated wart-infiltrating lymphocytes (WIL) from patients with clinically diagnosed anogenital warts. These WIL were characterized by their phenotype and their specificity for E7 and L1 proteins of HPV-6. The phenotype of WIL varied drastically from patient to patient, as determined by their expression of CD4, CD8, T-cell receptor alpha/beta chain (TCR alpha beta), and TCR gamma delta. Despite this heterogeneity in phenotype, HPV-6 E7 and/or L1-specific WIL, as determined by lymphoproliferation, could be isolated from more than 75% of the patients studied. Among all L1 peptides recognized by WIL, peptides 311-330 and 411-430 were the most consistently detected, with seven of nine patients for whom L1 peptide reactivity was observed responding to at least one of them. Moreover, the HPV-6 epitopic peptides recognized by WIL differed to some extent from those recognized by peripheral T cells.     

Complete genome sequence of canine papillomavirus type 9

Papillomaviruses are nonenveloped, double-stranded DNA viruses that are associated with both benign and malignant tumors in animals and humans. We report the complete genome sequence of canine papillomavirus type 9 isolated from a solitary pigmented plaque on a mixed-breed bloodhound.     

DNA sequence organization and transcription of the chicken genome

A new approach has been used to examine DNA sequence organization in the chicken genome. The interspersion pattern was determined by studying the fraction of labelled DNA fragments of different lengths that hybridized to an excess of short chicken repeated DNA sequences. The results indicate that chicken DNA has a pattern of sequence organization quite different than the standard ‘Xenopus’ or ‘Drosophila’ patterns. Two classes of unique sequences are found. One, 34% of the genome, consists of unique sequences approx. 4 kb long interspersed with repeated sequences. The second, non-interspersed fraction, 38% of the genome, consists of unique sequences found in long tracts, a minimum of approx. 22 kb in length. In an attempt to determine whether a relationship exists between DNA sequence organization and the distribution of structural genes we have isolated chicken DNA sequences belonging to different interspersion classes and tested each for the presence of structural genes by hybridization to excess poly(A)⁺ mRNA. Sequences complementary to poly(A)⁺ mRNA can be found with approximately the same frequency in both the non-interspersed fraction of the genome and a repeat-contiguous fraction enriched for interspersed sequences.     

Isolation and sequence organization of human ribosomal DNA.

The genes coding for 28 S and 18 S ribosomal RNA have been purified from leukemic leukocytes of one human individual by density gradient centrifugation. The purified ribosomal DNA was analyzed by restriction endonuclease digestion and electron microscopy. The location of cleavage sites for the restriction endonuclease EcoRI was established by R-loop mapping of restriction fragments by electron microscopy. The results are in agreement with gel analysis and gel transfer hybridization. One type of ribosomal DNA repeating unit contains four cleavage sites for EcoRI. Two of these cuts are located in the genes coding for 28 S and 18 S rRNA, while the other two are in the non-transcribed spacer. Thus, one of the restriction fragments generated contains non-transcribed spacer sequences only and is not detected by gel transfer hybridization if labeled rRNA is used as the hybridization probe. A second type of repeating unit lacks one of the EcoRI cleavage sites within the non-transcribed spacer. This indicates that sequence heterogeneity exists in human rDNA spacers. R-loop mapping of high molecular weight rDNA in the electron microscope reveals that the majority of repeats are rather uniform in length. The average size of 22 repeats was 43.65(±1.27) kb. Two repeats were found with lengths of 28.6 and 53.9 kb, respectively. This, and additional evidence from gels, indicates that some length heterogeneity does exist in the non-transcribed spacer. The structure of the human rDNA repeat is summarized in Figure 10.     

An electron microscope study of the DNA sequence organization of the human genome

The arrangements of inverted-repeated and repeated DNA sequences in the human genome have been investigated by an electron microscope method. The arrangement of the interspersed repeated DNA sequences is found to be similar to the corresponding arrangement found in Xenopus. This arrangement consists of 300-nucleotide-long repeated DNA sequences interspersed with roughly gene-size single-copy DNA sequences. The inverted-repeated sequences are also 300 nucleotides in length and are interspersed with the other DNA sequence classes.Most inverted-repeated sequences (64%) are spaced by another sequence which is recognized by electron microscopy as a single-stranded loop in a hairpin structure. The average length of this spacer loop is 1.6 kilobases. Although some pairs of inverted-repeated sequences are clustered, most seem to be randomly distributed throughout the genome. The average distance separating two pairs of inverted-repeated sequences is 10 to 20 kilobases. The interspersed repeated sequences and inverted-repeated sequences are arranged simultaneously in a portion of the human genome resulting in an interspersion of all three sequence classes.     

Nucleotide sequence and genome organization of bacteriophage S13 DNA

The complete sequence of bacteriophage S13 DNA has been determined. The molecule has 5386 nucleotides and differs from φX174 by 87 transitions and 24 transversions. All the proteins, A, A^*, B, C, D, E, F, G, H, J and K found in φX174 are also present in S13. Due to changes in the H/A intergenic region of S 13, the start of an additional protein. A′, has been identified. Genes F and H coding for the capsid and spike proteins, respectively, are the least conserved in comparison to φX174. Many of the silent changes, as well as some amino acid changes, are found in the same nucleotide sequence positions in phage G4, confirming the interrelationship between the three phages.     

DNA sequence organization in the genome of Cycas revoluta

The pattern of DNA sequence organization in the genome of Cycas revoluta was analyzed by DNA/DNA reassociation. Reassociation of 400 base pair (bp) fragments to various C₀t values indicates the presence of at least four kinetic classes: the foldback plus very highly repetitive sequences (15%), the fast repeats (24%), the slow repeats (44%), and the single copy (17%). The latter component reassociates with a rate constant 1×10^–4 M^–1S^–1 corresponding to a complexity of 1.6× 10⁶ kb per haploid genome. A haploid C. revoluta nucleus contains approximately 10.3 pg DNA. The single-copy sequences account for about 28% of the DNA, but only 17% reassociate with single-copy kinetics because of interspersion with repetitive sequences. — The interspersion of repetitive and single-copy sequences was examined by reassociation of DNA fragments of varying length to C₀t values of 70 and 500. A major (65%) and homogeneous class of single-copy sequences averaging 1,100 bp in length is interspersed in a short period pattern with repeated sequences. A minor (35%) heterogeneous single-copy component is interspersed in a long-period pattern. The majority of repetitive sequences have a length distribution of 100–350 bp with subclasses averaging 150 and 300 bp in length. Repeat sequences with a wide range in sizes exceeding 2 kilobase pair (kb) are also present in this genome. — The size and distribution of inverted repeat (ir) sequences in the DNA of C. revoluta were studied by electron microscopy. It is estimated that there are approximately 4 × 10⁶ ir pairs (one per 2.33 kb) that form almost equal numbers of looped and unlooped palindromes. This high value is 2.5 times that found in wheat DNA. These palindromes are in general randomly distributed in the genome with an average interpalindrome distance of 1.6 kb. The majority (about 85%) of ir sequences of both types of palindromes belong to a main-size class, with an average length of 210 bp in the unlooped and and 163 bp in the looped type. These values are comparable to those reported for some other plant and animal genomes. Distribution of length of single stranded loops showed a main-size class (75%) with an average length of 220 bp.     

DNA sequence organization in the genome of Nicotiana tabacum

The genome of Nicotiana tabacum was investigated by DNA/DNA reassociation for its spectrum of DNA repetition components and pattern of DNA sequence organization. The reassociation of 300 nucleotide DNA fragments analyzed by hydroxyapatite chromatography reveals the presence of three major classes of DNA differing in reiteration frequency. Each class of DNA was isolated and characterized with respect to kinetic homogeneity and thermal properties on melting. These measurements demonstrate that the genome of N. tabacum has a 1C DNA content of 1.65 pg and that DNA sequences are represented an average of 12,400, 252, and 1 times each. — The organization of the DNA sequences in the N. tabacum genome was determined from the reassociation kinetics of long DNA fragments as well as S1 nuclease resistance and hyperchromicity measurements on DNA fragments after annealing to C₀t values at which only repetitive DNA sequences will reassociate. At least 55% of the total DNA sequences are organized in a short period interspersion pattern consisting of an alternation of single copy sequences, averaging 1400 nucleotides, with short repetitive elements approximately 300 nucleotides in length. Another 25% of the genome contains long repetitive DNA sequences having a minimal genomic length of 1500 nucleotides. These repetitive DNA sequences are much less divergent than the short interspersed DNA sequence elements. These results indicate that the pattern of DNA sequence organization in the tobacco genome bears remarkable similarity to that found in the genomes of most animal species investigated to date.     

Genome organization and nucleotide sequence of human papillomavirus type 33, which is associated with cervical cancer.

The 7,909-nucleotide sequence of human papillomavirus type 33, which is associated with cervical cancer, has been determined and used to deduce the corresponding genome arrangement. Extensive sequence homologies and other genetic features are shared with the related oncogenic virus, human papillomavirus type 16, especially in the major reading frames. A surprising difference was found in the noncoding region of human papillomavirus type 33 as, unlike all other sequenced papillomaviruses, it contains a perfect 78-base pair tandem repeat.     

The DNA sequence of the human cytomegalovirus genome.

In the first part of this article we review what has been learnt from the analysis of the sequence of HCMV. A summary of this information is presented in the form of an updated map of the viral genome. HCMV is representative of a major lineage of herpesviruses distinct from previously sequenced members of this viral family and demonstrates striking differences in genetic content and organization. The virus encodes approximately 200 genes, including nine gene families, a large number of glycoprotein genes, and homologues of the human HLA class I and G protein-coupled receptor genes. The HCMV sequence thus provides a sound basis for future molecular studies of this highly complex eukaryotic virus. The second part discusses the practical rate of DNA sequencing as deduced from this and other studies. The 229 kilobase pair DNA genome of human cytomegalovirus (HCMV) strain AD169 is the largest contiguous sequence determined to date, and as such provides a realistic benchmark for assessing the practical rate of DNA sequencing as opposed to theoretical calculations which are usually much greater. The sequence was determined manually and we assess the impact of new developments in DNA sequencing.     

Size and structure of the bird genome. II. Repetitive DNA and sequence organization

1. Highly repetitive, middle repetitive and single copy DNA were evaluated in 19 species of birds, belonging to nine orders, by means of a reassociation kinetics method. 2. A rather uniform pattern is present in all the species studied (single copy = 60-75%; middle repetitive = 13-20% and highly repetitive 10-20%). 3. Reassociation kinetics of fragments of different length confirms the presence of a long period interspersion pattern. 4. Among different orders, no significant differences are observed. 5. DNA sequence organization seems to be related to genome size, with an inverse correlation between DNA nuclear content and amount of interspersed repetitive sequences.     

Human papillomavirus type 6 long control region and human cellular DNA contain related sequences.

We have identified a region of human papillomavirus type 6 (HPV-6) DNA that hybridizes with human cellular DNA containing no detectable HPV DNA sequences. The region of hybridization has been localized to a segment of the viral long control region between the end of the L1 open reading frame and the late polyadenylation signal and is likely contained within a 94-base-pair insertion at nucleotide 7350 which is present in the cloned HPV-6b DNA used for these studies. Restriction fragments of HPV-6 DNA from seven patients suggested that this insert was present in these naturally occurring viral genomes as well. The presence of this insert was confirmed by direct sequence analysis of polymerase chain reaction-amplified segments from four naturally occurring HPV-6 genomes. By analogy with other systems, this insert and surrounding sequences may function to destabilize the HPV-6 late mRNA.     

Gene distribution and nucleotide sequence organization in the human genome

Human DNA was fractionated by centrifugation in Cs2SO4 density gradients containing 3,6-bis(acetatomercurimethyl)dioxane (BAMD). Fractions were investigated in their analytical CsCl profiles and a number of specific sequences were localized in them. The results so obtained led to an improved understanding of the organization of nucleotide sequences in the human genome, as well as to the discovery that a class of DNA having a very high G + C content and not represented in the mouse genome, is particularly rich in genes and interspersed repetitive sequences.     

Biologic properties and nucleotide sequence analysis of human papillomavirus type 51.

Human papillomaviruses (HPVs) may be grouped according to the site from which they are isolated and the disease with which they are associated. We recently identified and cloned HPV type 51 (HPV-51) from a low-grade precancerous lesion (G. Nuovo, E. DeVilliers, R. Levine, S. Silverstein, and C. Crum. J. Virol. 62:1452-1455, 1988). Molecular epidemiologic analysis of cervical lesions, including condylomata and low- and high-grade precancers, revealed that HPV-51 was present in about 5% of the samples we examined. We have now determined the complete nucleotide sequence of this virus and compared it with other sequenced HPVs. Our analysis reveals that the 7,808-bp genome is composed of eight open reading frames which are encoded on the same strand and that this virus is most closely related to HPV-31. Sequence comparisons place this virus in the group of high-risk viruses (those with an increased risk of progressing to malignancy) along with HPV-16, -18, -31, and -33. Morphologic transformation experiments demonstrated that HPV-51 had transformation potential and that transformed cells contained RNAs homologous to E6 and E7.     

Nucleotide sequence and genome organization of canine parvovirus.

The genome of a canine parvovirus isolate strain (CPV-N) was cloned, and the DNA sequence was determined. The entire genome, including ends, was 5,323 nucleotides in length. The terminal repeat at the 3' end of the genome shared similar structural characteristics but limited homology with the rodent parvoviruses. The 5' terminal repeat was not detected in any of the clones. Instead, a region of DNA starting near the capsid gene stop codon and extending 248 base pairs into the coding region had been duplicated and inserted 75 base pairs downstream from the poly(A) addition site. Consensus sequences for the 5' donor and 3' acceptor sites as well as promotors and poly(A) addition sites were identified and compared with the available information on related parvoviruses. The genomic organization of CPV-N is similar to that of feline parvovirus (FPV) in that there are two major open reading frames (668 and 722 amino acids) in the plus strand (mRNA polarity). Both coding domains are in the same frame, and no significant open reading frames were apparent in any of the other frames of both minus and plus DNA strands. The nucleotide and amino acid homologies of the capsid genes between CPV-N and FPV were 98 and 99%, respectively. In contrast, the nucleotide and amino acid homologies of the capsid genes for CPV-N and CPV-b (S. Rhode III, J. Virol. 54:630-633, 1985) were 95 and 98%, respectively. These results indicate that very few nucleotide or amino acid changes differentiate the antigenic and host range specificity of FPV and CPV.