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The Spanish influenza pandemic of 1918-1919 caused acute illness in 25-30% of the world's population and resulted in the death of 40 million people. The complete genomic sequence of the 1918 influenza virus will be deduced using fixed and frozen tissues of 1918 influenza victims. Sequence and phylogenetic analyses of the complete 1918 haemagglutinin (HA) and neuraminidase (NA) genes show them to be the most avian-like of mammalian sequences and support the hypothesis that the pandemic virus contained surface protein-encoding genes derived from an avian influenza strain and that the 1918 virus is very similar to the common ancestor of human and classical swine H1N1 influenza strains. Neither the 1918 HA genes nor the NA genes possessed mutations that are known to increase tissue tropicity, which accounts for the virulence of other influenza strains such as A/WSN/33 or fowl plague viruses. The complete sequence of the nonstructural (NS) gene segment of the 1918 virus was deduced and tested for the hypothesis that the enhanced virulence in 1918 could have been due to type I interferon inhibition by the NS1 protein. The results from these experiments were inconclusive. Sequence analysis of the 1918 pandemic influenza virus is allowing us to test hypotheses as to the origin and virulence of this strain. This information should help to elucidate how pandemic influenza strains emerge and what genetic features contribute to their virulence.

The influenza A virus pandemic of 1918-1919 resulted in an estimated 20-40 million deaths worldwide. The hemagglutinin and neuraminidase sequences of the 1918 virus were previously determined. We here report the sequence of the A/Brevig Mission/1/18 (H1N1) virus nonstructural (NS) segment encoding two proteins, NS1 and nuclear export protein. Phylogenetically, these genes appear to be close to the common ancestor of subsequent human and classical swine strain NS genes. Recently, the influenza A virus NS1 protein was shown to be a type I IFN antagonist that plays an important role in viral pathogenesis. By using the recently developed technique of generating influenza A viruses entirely from cloned cDNAs, the hypothesis that the 1918 virus NS1 gene played a role in virulence was tested in a mouse model. In a BSL3+ laboratory, viruses were generated that possessed either the 1918 NS1 gene alone or the entire 1918 NS segment in a background of influenza A/WSN/33 (H1N1), a mouse-adapted virus derived from a human influenza strain first isolated in 1933. These 1918 NS viruses replicated well in tissue culture but were attenuated in mice as compared with the isogenic control viruses. This attenuation in mice may be related to the human origin of the 1918 NS1 gene. These results suggest that interaction of the NS1 protein with host-cell factors plays a significant role in viral pathogenesis.

Phylogenetically informative amino acid positions (PIPs) were identified in influenza A neuraminidases of subtypes N1 and N2. Neuraminidase evolves in a lineage-specific way as the virus adapts to a new host or changes to evade the host's immune system. Thus, many PIPs undoubtedly identify positions involved in virus-host interactions. Phylogenetically important regions (PIRs) are defined as several PIPs near one another. There are 15 PIRs on N1 and 12 on N2, seven of which are shared between the two subtypes. Many PIRs are coincident with antigenic or glycosylation sites. Other PIRs may represent additional antigenic sites or may be involved in other aspects of virus-host biology., (Copyright 2000 Academic Press.)

The "Spanish" influenza pandemic of 1918 was characterized by exceptionally high mortality, especially among young adults. The surface proteins of influenza viruses, hemagglutinin and neuraminidase, play important roles in virulence, host specificity, and the human immune response. The complete coding sequence of hemagglutinin was reported last year. This laboratory has now determined the complete coding sequence of the neuraminidase gene of the 1918 virus. Influenza RNA fragments were isolated from lung tissue of three victims of the 1918 flu; complete sequence was generated from A/Brevig Mission/1/18, with confirmatory sequencing carried out on A/South Carolina/1/18 and A/New York/1/18. The 1918 neuraminidase gene sequence was compared with other N1 subtype neuraminidase genes, including 9 N1 strains newly sequenced for this study. The 1918 neuraminidase shares many sequence and structural characteristics with avian strains, including the conserved active site, wild-type stalk length, glycosylation sites, and antigenic sites. Phylogenetically, the 1918 neuraminidase gene appears to be intermediate between mammals and birds, suggesting that it was introduced into mammals just before the 1918 pandemic.

A long-finned pilot whale with morbilliviral disease was stranded in New Jersey. An immunohistochemical stain demonstrated morbilliviral antigen. Reverse transcriptase-polymerase chain reaction for morbillivirus P and N genes was positive. Novel sequences most closely related to, but distinct from, those of dolphin and porpoise morbilliviruses suggest that this virus may represent a third member of the cetacean morbillivirus group.

A parsimony approach was used to construct phylogenetic trees of the H1, H2 and H3 influenza hemagglutinin subtypes. The parsimony trees were then compared with randomly generated trees to identify regions of the proteins containing the most phylogenetic information, i.e. those regions making the parsimony trees shorter. We reasoned that any areas of the hemagglutinin protein that were phylogenetically 'information-rich' would be good candidates for sites involved in virus-host interactions and their identification might lead to a better understanding of the protein. Molecular modelling, based upon the crystal structure of the H3 hemagglutinin, demonstrated that most phylogenetically important regions of the H1 subtype were on the surface of the hemagglutinin trimer, primarily in the globular region. Many corresponded to known antigenic or receptor binding sites, while others appear to be novel and specific for H1.

The published C-terminal sequence of Escherichia coli 50S ribosomal protein L31, ellipsisRFNK (Brosius, J. (1978) Biochemistry 17, 501-508), differs from that predicted by the gene sequence, ellipsisRFNKRFNIPGSK (GenBank accession no. X78541). This discrepancy might be due to post-translational processing of the protein. To examine this possibility, we have isolated L31 from E. coli strain MRE600 and sequenced the C-terminal tryptic peptide. We find the sequence to be FBIPGSK. Size comparisons of L31 from several E. coli strains demonstrate that all are identical in size to the protein isolated from MRE600 and larger than the previously described protein, indicating that ellipsisRFNKRFNIPGSK represents the true C-terminus of L31. In addition, we show that the failure to identify L31 in many ribosome preparations is probably due to the protein's loose association with the ribosome and its ability to form various intramolecular disulfide bonds, leading to L31 forms with distinct mobilities in gels.

The "Spanish" influenza pandemic killed over 20 million people in 1918 and 1919, making it the worst infectious pandemic in history. Here, we report the complete sequence of the hemagglutinin (HA) gene of the 1918 virus. Influenza RNA for the analysis was isolated from a formalin-fixed, paraffin-embedded lung tissue sample prepared during the autopsy of a victim of the influenza pandemic in 1918. Influenza RNA was also isolated from lung tissue samples from two additional victims of the lethal 1918 influenza: one formalin-fixed, paraffin-embedded sample and one frozen sample obtained by in situ biopsy of the lung of a victim buried in permafrost since 1918. The complete coding sequence of the A/South Carolina/1/18 HA gene was obtained. The HA1 domain sequence was confirmed by using the two additional isolates (A/New York/1/18 and A/Brevig Mission/1/18). The sequences show little variation. Phylogenetic analyses suggest that the 1918 virus HA gene, although more closely related to avian strains than any other mammalian sequence, is mammalian and may have been adapting in humans before 1918.

The "Spanish" influenza pandemic killed at least 20 million people in 1918-1919, making it the worst infectious pandemic in history. Understanding the origins of the 1918 virus and the basis for its exceptional virulence may aid in the prediction of future influenza pandemics. RNA from a victim of the 1918 pandemic was isolated from a formalin-fixed, paraffin-embedded, lung tissue sample. Nine fragments of viral RNA were sequenced from the coding regions of hemagglutinin, neuraminidase, nucleoprotein, matrix protein 1, and matrix protein 2. The sequences are consistent with a novel H1N1 influenza A virus that belongs to the subgroup of strains that infect humans and swine, not the avian subgroup.

Chromosomal studies in three species of Amazonian Callithrix (2n=44) and data in the literature show that this group is karyomonotypic. Moreover, it is characterized by the presence of abundant heterochromatic regions, unlike the situation in congeneric forms of Callithrix of the Atlantic coast with 2n=46, and by the presence of a highly repetitive, exclusive DNA component, with a basic repeat motif of 1528bp. Karyotypic comparisons with other Callitrichids and an outgroup species showed that Callitrichids are karyologically conserved and explained several rearrangements that had presumably occurred during their phyletic radiation. Analyses of karyologic data enabled the construction of two alternative phylogenetic topologies. The lack of derived homoeologies, common to all members of the genus Callithrix grouped at present, and the fact that Amazonian species were more similar to Cebuella pygmaea (2n=44) than to their congeneric forms with 2n=46 suggested that species at present included in the Amazonian Callithrix should be grouped with C. pygmaea.

Human LINE-1 (L1Hs) retrotransposons can act as insertional mutagens and are expressed in a variety of tumors, including breast cancer. The purpose of the present study was to examine expression of the p40 protein encoded by the first open reading frame of a L1Hs element in normal human breast tissue of patients without malignant breast disease and in nontumor breast tissue adjacent to cancer and to compare it to expression in breast carcinomas. An antiserum specific for the L1Hs p40 protein was used to analyze its expression in 5 reduction mammoplasties, 16 primary breast cancers, 1 lymph node metastasis and 13 non-malignant breast tissues adjacent to matched primaries by western blotting and/or immunocytochemistry. The immunoreactive band observed on westerns consistently had a M(r) of approximately 46 kDa. Westerns detected some p40 protein expression in all malignant and nonmalignant tissues examined, although 4 of 5 reduction mammoplasties had very low or trace levels as compared with tumors. Nonmalignant breast tissues adjacent to cancers showed significant western band reactivity, and all 15 tumors were positive. Immunocytochemistry revealed staining specificity of the antibody for epithelial cells. Of 12 invasive carcinomas examined, 100% were positive for the p40 protein, whereas one reduction with benign proliferative disease was very weakly reactive, two histologically normal reductions were negative, and 4 of 6 nonmalignant tissues adjacent to cancers were negative. Our data indicated that expression of the L1Hs p40 protein was often elevated in tumor cells of human breast cancers compared to epithelium of normal mammary gland.

The phylogenetic relationships of 56 nuclear hormone receptors from both invertebrates and vertebrates were determined by the parsimony method (PAUP). The consensus tree suggests that the ancestral gene diverged into five major subfamilies, each of which evolved into at least one cluster of related molecules. These subfamilies are represented by: (1) thyroid hormone receptors (TR); (ii) steroid receptors (SR); (iii) retinoic acid receptors (RAR), retinoid X receptors (RXR), and the chicken ovalbumin upstream promoter transcription factor 1 (COUP) group; (ix) peroxisome proliferator-activated receptors (PPAR); and (v) vitamin D receptor (VDR) and knirps (kni) group. Although the neighbor-joining (N-J) method clustered the receptors into a greater number of subfamilies, it was evident that the components of the terminal receptor subgroups were similar to those found in the PAUP tree. These terminal clusters might then represent phylogenetically stable relationships. The positions of some orphan receptors were perturbed when a different algorithm was employed in the analysis. Both PAUP and N-J evolutionary trees showed that the receptors within the subgroups of a major sublineage tend to recognize hormones of very similar structure. This finding suggests that the relative phylogenetic position of orphans to well-characterized receptors might be exploited to predict the type of ligand they would recognize.

Two satellite DNAs, designated CapA and CapB, were isolated from the neotropical primate, Cebus apella. The satellites exhibit nonoverlapping distributions on C. apella chromosomes. CapA is a major component of interstitial regions of constitutive heterochromatin, a very large block of heterochromatin comprising most of the long arm of chromosome 11, and some telomeres. The CapA monomer has a length of about 1500 bp and appears recently to have undergone an amplification episode in the C. apella genome. CapA-like sequences are probably present in members of the family Cebidae (to which C. apella belongs), but not in members of the family Callitrichidae (marmosets). CapB sequences can be detected at the centromeres of many C. apella chromosomes, and similar sequences are present in all neotropical primates. The 342 bp CapB monomer shares 60%-64% sequence identity with several alpha satellite sequences of human origin. Because of its structure, sequence, and location, it appears that CapB is the New World primate homolog of Old World primate alpha satellite DNA.

An antibody to the protein encoded by the first open reading frame of the human LINE-1 (L1Hs) element was used to examine immunohistochemically 59 formalin-fixed, human testicular germ cell tumors. Six tumors were positive for L1Hs expression. In all cases the L1Hs-positive cells were epithelial and most had the very characteristic, undifferentiated appearance of embryonal carcinoma or yolk sac tumor cells. One L1Hs-positive tumor had metastasized to the lung and lymph nodes and the metastatic cells also expressed L1Hs. This is the first observation of widespread retrotransposon expression in human tissue. These observations raise the possibility that L1Hs-encoded proteins may function as oncoproteins in some cancers.

The major centromeric satellite nt sequences present in the domestic dog (Canis familiaris) and in the grey fox (Urocyon cineroargenteus) have been examined. The dog satellite monomer is 737 bp long and contains 51% G + C; the grey fox satellite monomer is 880 b long and contains 54% G + C. The two satellites share three regions of 78, 92 and 314 bp with 70-80% sequence similarity. Sequence data from 16 monomers of dog satellite and 19 monomers of grey fox satellite demonstrate that the substitution spectra are different in the two canid species. For example, substitutions involving G or C residues are much more common in the grey fox satellite than in the domestic dog satellite despite their similar G + C contents.

5S RNA-protein complexes were prepared in vitro using partially purified E. coli 5S RNA and total E. coli 70S ribosomal proteins. The complexes were isolated from sucrose gradients and shown to contain proteins L5, L18, L25 and a fourth protein not heretofore characterized and designed L31. The complexes were treated with the crosslinking reagents dimethyl suberimidate and dimethyl-3,3'-dithiobispropionimidate. Both reagents gave identical patterns of crosslinked proteins when analyzed by one-dimensional polyacrylamide/dodecylsulfate gel electrophoresis. Dimers of L5-L31', L5-L18 and L18-L18 and a trimer containing L5, L18 and L31' were identified by diagonal polyacrylamide/dodecylsulfate gel electrophoresis of the proteins crosslinked with dimethyl-3,3'-dithiobispropionimidate. No crosslinking was detected between L25 and the other three proteins.

Restriction mapping demonstrated the presence of several distinct proviral forms of mouse mammary tumor virus in the genome of GR mice. One of these proviruses (GR-MTV-2) was highly amplified in GR 3A cells, a cell line derived from a GR mammary tumor. By the criterion of restriction mapping, the amplified GR-MTV-2 provirus found in GR 3A cells was identical to the provirus found in M1.19 cells, a rat cell line infected with virions obtained from GR 3A culture fluid. This result clearly implies that the GR-MTV-2 provirus in GR 3A cells was transcribed into the virion-associated viral RNA genome. Cleavage of either GR 3A or M1.19 cell DNAs with the restriction enzyme Bg1 II gave rise to a 2.6 x 10(6) dalton GR-MTV-2 proviral fragment (ca. 45% of the viral genome). This fragment was isolated and mapped with thirteen restriction enzymes.

Iodination of Escherichia coli ribosomal protein L18 inactivated the 5 S RNA binding activity of the protein. Complete activity loss occurred at a 4-fold molar excess of iodine to L18. Tyrosine was found to be the reactive amino acid. L18, prebound to 5 S RNA, was inactivated at a much slower rate than unbound L18. Treatment of L18 with tetranitromethane also resulted in an inactivation of the protein. However, much larger amounts of tetranitromethane, compared to iodine, were necessary to achieve inactivation (50% activity loss at a 600-fold molar excess of tetranitromethane to L18).

A large proportion (0.5-1%) of total mouse DNA is cleaved by Bam HI into fragments whose size is about 500 base pairs. A cloned member of this repetitive family of DNA sequences (BAM5 family) was sequenced by the dideoxy chain termination procedure and shown to contain 507 base pairs. The sequence exhibited no unusual or remarkable features. Repetitive sequences complementary to the cloned BAM5 fragment were found in rat DNA, but not in feline or human DNA. Restriction mapping suggested that many BAM5 sequences were components of much larger repetitive DNAs which were scattered throughout the mouse genome. The BAM5 sequences within the larger repetitive DNAs did not appear to be arranged tandemly or as members of scrambled tandem repeats. RNA homologous to the cloned BAM5 sequence was detected in cultured mouse cells, but not in cultured rat cells.

Callimico goeldii (Goeldi's marmoset) is a neotropical primate with 2n = 47,X1X2Y in the male, and 2n = 48,X1X1X2X2 in the female, due to a Y-autosome translocation. Karyological comparisons of Callimico, Callithrix jacchus and Cebus apella suggest that Callimico is a member of the Callitrichidae. Isozyme data and restriction mapping of LINE-1 repetitive elements in these species and in a variety of other neotropical primates confirm these findings and supply strong evidence for including Callimico in the Callitrichidae.

The KpnI and BamH1 (or M1F-1) families are the predominant sets of long interspersed repeated DNA sequences (LINEs) in primates and rodents, respectively. Recently, the sequences of several cloned subsegments from each family were determined in different laboratories. These sequences have now been compared and found to be homologous over at least 1400 bp. The data suggest that the two LINE families had a common progenitor and have been conserved in similar abundance although in divergent forms in the two mammalian orders.

A major satellite DNA has been cloned from the domestic cat (Felis catus) and characterized. The satellite monomer, termed FA-SAT, is 483 base-pairs in size, 64% G + C, and represents about 1 to 2% of the cat genome. A consensus sequence based upon partial sequence data from 21 independently isolated clones demonstrates: (1) FA-SAT is not composed of a series of shorter repeats, although about 25 copies, primarily imperfect, of the hexanucleotide TAACCC appear in the sequence; (2) there are many more CpG dinucleotides present in FA-SAT than expected for a random sequence of its size; and (3) 61% of all base substitutions in FA-SAT involve the replacement of G and C residues by A and T residues, indicating that FA-SAT is rapidly becoming A + T-rich. FA-SAT-related sequences are found in many mammals, where they appear to be scattered throughout the genome and not tandemly arranged as in the cat. An FA-SAT-related sequence was cloned from the domestic dog genome and sequenced, and shown to contain multiple copies of the same TAACCC hexanucleotide found in the cat satellite.

The BALB/c (C-) mouse hyperplastic outgrowth line (D1) was used to study murine mammary tumor virus (MuMTV) expression in both D1 and tumors derived from D1. D1 was transplanted into virus-infected BALB/cfC3H (C+) and virus-uninfected C- animals. In duplicate studies, tumor incidence was the same in both groups. However, the tumor latency period was longer for D1 transplanted into C+ mice (D1/C+) than for D1 transplanted into C- mice (D1/C-). MuMTV was detected in 85% of D1/C+ outgrowths and in 29% of D1/C+ tumors but was never detected in D1/C- outgrowths or tumors. D1/C- outgrowth and tumors and most of the D1/C+ tissues expressed little or no MuMTV RNA. Some D1/C+ tumors expressed substantial levels of MuMTV RNA. These same tumors also had MuMTV antigen and contained the exogenously acquired C3H-MuMTV provirus in the cellular DNA as shown by DNA fragment patterns following cleavage with Pst I and Eco RI endonucleases. D1/C+ tumors containing no viral RNA were also antigen-negative and lacked the acquired C3H provirus. These studies indicate that D1 has substantially changed in its incidence and in its response to MuMTV. MuMTV infection was not tumorigenic in the traditional sense, a finding that has led to a reevaluation of our current models of virus-host relationships and the biology of precancerous conditions.

The BAM HI family of long interspersed DNAs in mice represent as much as 0.5% of the mouse genome. Cloned mouse DNA fragments which contain BAM HI/non-BAM HI junction sequences have been analyzed by restriction mapping and DNA sequencing. It has been found that BAM HI elements: (i) are approximately 7 kilobase pairs in size, (ii) are not bracketed by long repeated sequences analogous to the terminal repeats of proviruses and (iii) contain a poly-dA track at one end. The data strongly suggest that BAM HI elements arose by a process involving RNA intermediates. The beginning of the element, opposite the poly-dA track, contains a 22 base pair sequence exhibiting 65% homology to a ubiquitous mammalian sequence which may play a role in DNA replication (1). The poly-dA end of the element contains BAM5 and R sequences, both of which have been described previously (2,3).

The DNA's isolated from five mouse hyperplastic mammary gland outgrowth lines from BALB/cfC3H mice were digested with the restriction endonucleases PsTI, BamHI, or EcoRI; electrophoresed; and analyzed by Southern blotting and autoradiography. Proviral DNA sequences from the acquired C3H mouse mammary tumor virus were detected in the DNA of all five lines, indicating that they were infected. The DNA of the five hyperplastic lines contained more EcoRI and BamHI mouse mammary tumor virus proviral DNA fragments than did DNA from normal organs, suggesting that the hyperplastic tissues were composed of more homogeneous cell populations than was lactating mammary gland. Each hyperplastic line had unique and reproducible BamHI and EcoRI restriction (integration) patterns which were stable over as many as seven transplant generations. Three sublines, which originated from the same hyperplastic alveolar nodule, had unique integration patterns but also shared several fragments. On the basis of these observations, we propose that mouse mammary "hyperplasias" are clonal dominant premalignant neoplasms.

Two families of tandemly repeated satellite DNAs were isolated from the neotropical primate Callimico goeldii (Goeldi's marmoset). One satellite, CgoA, is over 70% A + T and has a monomer length of 338 bp. The other satellite, CgoB, is 50% A + T and has a monomer length of 916 bp. Both CgoA and CgoB hybridize strongly with Callimico DNA, but not with the DNA of other new and old world primates. Based upon a neutral substitution rate of 1.5 X 10(-9)/site per year for primates, sequence data from 15 CgoA monomers indicate that the tandem array is at least 30 million years old. Since no other neotropical primate has amplified CgoA sequences, the data suggest that the ancestor of Callimico separated from the other neotropical primates at least 30 million years ago. This value is about fourfold larger than the value of 7-9 million years derived from immunological data by Sarich and Cronin (1980). Possible reasons for this discrepancy are discussed.

In a previous publication1 we reported that the tyrosine selective reagent, tetraitromethane, causes complete inactivation of E. coli 30S ribosomes for poly U directed non-enzymatic phe-tRNA binding. This inactivation was demonstrated to be due to the chemical modification of the protein moiety of the ribosome. We have no identified the proteins of the 30S particle inactivated by this modification. Using a method of ribosome reconstruction we have found that unmodified proteins S1, S11, and S21 are essential for the restoration of the phe-tRNA binding activity of tetranitromethane inactivated ribosomes. We propose that these three proteins are intimately involved in the 30S ribosome binding site for tRNA.