Molecular cloning of a pea H1 histone cDNA

A pea (Pisum sativum, var. Little Marvel) H1 histone cDNA has been isolated from a lambda gt11 expression vector library. This cDNA has been sequenced and shown to represent the entire protein-coding region of the mRNA. The deduced protein sequence is 265 amino acids long (28018 Da) and contains 70 lysines and 3 arginines. The structure of the encoded protein is comparable to animal lysine-rich histones. The central region, which has an amino acid composition similar to that found in the globular domains of animal lysine-rich histones, is flanked by an amino-terminal region rich in lysine, glutamic acid and proline and by a carboxyl-terminal region rich in lysine, alanine, valine and proline. Despite the structural similarities, the protein has little sequence homology with animal lysine-rich histones. This H1 protein is unusual because 12 of the first 40 amino acids are glutamic acid.     

Sequence of the amino terminal half of rat testis-specific histone variant H1t

H1t is a testis-specific H1 histone variant that appears during the meiotic phase of spermatogenesis in mammals. The sequence of the first 108 residues of rat H1t have been determined and compared to boar H1t and also to a consensus sequence characteristic of standard somatic H1 variants. The two versions of H1t share a common pattern of divergence from the standard somatic consensus. For example, they both share many substitutions within the H1 globular region (residues 40-110), a portion of the molecule that is virtually invariant among the common somatic variants. Within the entire region sequenced, there are 28 shared locations where both forms of H1t differ from the somatic consensus. However, identical substitutions occur at only 15 of these sites, and each protein also differs from the consensus at five (boar) or ten (rat) additional locations that are not shared. These results establish that H1t from diverse sources shows a characteristic pattern of divergence from the sequence of standard somatic H1 proteins. However, it is also clear that there is great tolerance for species-specific variation within this H1 class.     

Human spleen histone H1. Isolation and amino acid sequences of three minor variants, H1a, H1c, and H1d

Following the previous determination of the main variant H1b of human spleen histone H1, we have determined the complete amino acid sequence of another variant, H1d. Limited chymotryptic digestion of H1d produced four fragments, I to IV, and one partial fragment I-II, as in the case of H1b. These fragments were aligned with two overlapping peptides, produced by another enzyme from the intact H1d. We also confirmed the C-terminal sequence of H1d by carboxypeptidase digestion. This H1d has an acetylated N-terminal serine, equimolar alanine or valine residue at 17, and is composed of 212 residues. The molecular weight was 21,233 for the alanine variant and 21,261 for the valine variant in the unmodified form. We also deduced the total sequences of H1a and H1c in a similar way, considering the maximum homology with H1b and H1d. Each N-terminal serine residue is acetylated, too. H1a consists of 222 amino acid residues and has a molecular weight of 22,178 in its unmodified form; the H1c consists of 220 residues and has a molecular weight of 22,218 in that form. The human spleen H1 sequences varied to about the same extent in the N-terminal 40 and C-terminal 110 residues. However, the sequences of the about 70 internal residues are well conserved between the variants. The extent of differences among the human H1 variants is similar to, or rather smaller than, those among the mammalian somatic H1 species. The implications of these differences in the sequence for H1 function are discussed from the evolutionary viewpoint.     

Characterization of the chicken histone H1 gene complement. Generation of a complete set of vertebrate H1 protein sequences

Sequence analysis of four chicken H1 histone genes described here completes the characterization of the full complement of six H1 genes in the chicken genome. Each of the six genes codes for a different H1 protein sequence, and these range in size from 217 to 224 amino acids. The proteins are distinct in sequence from the H1-related chicken H5 protein and appear to be analogous to the standard somatic mammalian H1 subtypes. The protein sequence data deduced from the genes represent the first complete set of vertebrate H1 protein sequences. Comparison of the chicken H1 gene noncoding sequences with each other and with H1 gene sequences from other organisms reveals conservation of an H1 gene-specific element, a G-rich element, and histone gene-specific 3' elements. Additional sequences are conserved between H1 genes of the chicken and other vertebrates. Comparisons also reveal variation in promoter and 3' elements between chicken genes that could play a role in the differential expression of H1 gene protein products.     

The carboxyl-terminal domain of murine H1(0). Immunochemical and partial amino acid sequence comparisons with other H1(0)/H1/H5 histones

The carboxyl-terminal domain of murine H1(0) histone was compared with that of human H1(0), bovine H1(0) and other H1 and H5 histones. Two sets of antibodies were induced by murine H1(0). One set reacted with only the carboxyl-terminal domain of murine H1(0) and preferred the murine over the bovine and human proteins. The second set of antibodies reacted with the globular domain of murine H1(0) and did not distinguish among murine, bovine and human H1(0) species. There were five positions in the first 60 residues of the carboxyl-terminal domain in which the murine H1(0) differed from the human H1(0). In this region, the murine H1(0) had no more than 49% overall homology with other H1 and H5 histones; however, short sequences in the domain were very similar to short sequences that occur in rabbit H1.3, trout H1 and goose or chicken H5. In comparisons based on these and other published data, the carboxyl-terminal domain of H1(0) is found to be more variable among species than is the globular domain; the first two-thirds of the H1(0) carboxyl-terminal domain is largely unique and does not show great overall homology with H1 or H5, whereas the last third is again more conserved. As the first two-thirds of the domain is the only portion where the homology with H5 is less than 50%, it may be responsible for functional differences between H1(0) and H5.     

Nucleotide sequence of rat alpha 1-acid glycoprotein messenger RNA

The complete nucleotide sequence of rat alpha 1-acid glycoprotein (alpha 1-AGP) mRNA has been determined from cloned double-stranded cDNA. The coding portion of the mRNA was bounded at the ends by a 5'-untranslated region of 35 nucleotides in length and a 3'-untranslated region of 119 nucleotides in length. The 3'-untranslated region contains the characteristic AAUAAA sequence ending 18 nucleotides from the 3'-terminal poly(A) segment. The 5'-region of the mRNA contains two in-phase AUG codons separated by 12 nucleotides. Comparison with the known NH2-terminal amino acid sequence of serum rat alpha 1-AGP suggests that the primary translation product of the mRNA contains an additional 14 or 18 amino acids that are not present in the mature form of the protein, which contains 187 amino acids. The inferred amino acid sequence of rat alpha 1-AGP and the known amino acid sequence of human alpha 1-AGP have several regions of identity clustered in the NH2-terminal portion of the proteins. The carboxyl-terminal regions show significantly less homology. Six potential asparagine glycosylation sites are found in the rat sequence, and four of these sites are in positions similar to known glycosylation sites in the human protein. Furthermore, three of these potential glycosylation sites are in a region that exhibits extensive amino acid sequence conservation, suggesting that this region may be important for the biological function of alpha 1-AGP.     

Fine mapping of a cis-acting sequence element in yellow fever virus RNA that is required for RNA replication and cyclization

We present fine mapping of a cis-acting nucleotide sequence found in the 5' region of yellow fever virus genomic RNA that is required for RNA replication. There is evidence that this sequence interacts with a complementary sequence in the 3' region of the genome to cyclize the RNA. Replicons were constructed that had various deletions in the 5' region encoding the capsid protein and were tested for their ability to replicate. We found that a sequence of 18 nucleotides (residues 146 to 163 of the yellow fever virus genome, which encode amino acids 9 to 14 of the capsid protein) is essential for replication of the yellow fever virus replicon and that a slightly longer sequence of 21 nucleotides (residues 146 to 166, encoding amino acids 9 to 15) is required for full replication. This region is larger than the core sequence of 8 nucleotides conserved among all mosquito-borne flaviviruses and contains instead the entire sequence previously proposed to be involved in cyclization of yellow fever virus RNA.     

Definition of Amino Acid Residues on the Epitope Responsible for Recognition by Influenza A Virus H1-Specific, H2-Specific, and H1- and H2-Cross-Reactive Murine Cytotoxic T-Lymphocyte Clones

We defined the epitopes recognized by three influenza A virus-specific, H-2K^d-restricted CD8⁺ cytotoxic T-lymphocyte (CTL) clones: H1-specific clone A-12, H2-specific clone F-4, and H1- and H2-cross-reactive clone B7-B7. The A-12 and B7-B7 clones recognized the same peptide, which comprises amino acids 533 to 541 (IYSTVASSL) of A/PR/8 hemagglutinin (HA). The F-4 and B7-B7 clones both recognized the peptide which comprise amino acids 529 to 537 (IYATVAGSL) of A/Jap HA. Amino acids 533 to 541 of A/PR/8 HA are compatible with amino acids 529 to 537 of A/Jap HA. Amino acid S at positions 3 and 7 was responsible for recognition by H1-specific clone A-12, while amino acid G at position 7 was responsible for recognition by H2-specific clone F-4. Two conserved amino acids, T at position 4 and A at position 6, were responsible for recognition by H1-, and H2-cross-reactive clone B7-B7. These results indicate that a single nine-amino-acid region is recognized by HA-specific CTL clones of three different subtype specificities and that the amino acids responsible for the recognition by the CTL clones are different.     

The primary structure of rat liver glycogen synthase deduced by cDNA cloning. Absence of phosphorylation sites 1a and 1b

The cDNA for rat liver glycogen synthase was isolated by screening a rat liver cDNA library constructed in lambda gt11. The cDNA was 2.4 kilobases in length and encoded a protein of 703 amino acid residues with a molecular mass of 80.5 kDa. Comparison of the rat liver and the human muscle sequences show that the amino- and carboxyl-terminal regions are quite divergent as compared to the internal sequences which show an 80% identity. The rat liver carboxyl-terminal region is truncated by 33 residues and has only 46% identity with the muscle sequence but retains the common feature of a low content of hydrophobic amino acids (13%). Phosphorylation sites 1a and 1b, which are the primary targets for phosphorylation by cAMP-dependent protein kinase, are absent in the liver sequence. The presence of these divergent, structurally anomalous carboxyl-terminal regions in liver and muscle glycogen synthase suggests the absence of the requirement that they possess a tertiary structure that is integral to that of the protein core. A model is proposed in which this region interacts with a catalytic core to maintain the I state, and in which phosphorylation serves to uncouple this interaction.     

The primary structure of the major isoform (H1.1) of histone H1 from the nematode Caenorhabditis elegans.

The complete primary structure of the major isoform (H1.1) of histone H1 from the nematode Caenorhabditis elegans was determined. The amino acid chain consists of 207 amino acids and has a blocked N-terminus. The nematode histone shows rather little sequence identity when compared with proteins of the H1 family derived from other organisms. However, the main characteristic features of H1 molecules have been well conserved: a tripartite domain structure consisting of a central hydrophobic core of about 80 residues, flanked by an N-terminal domain which is somewhat acidic at the very N-terminus, but very basic further on, and a long C-terminal domain very rich in lysine, alanine and proline. Several repeat structures, including a twice (with modification)-repeated and well-conserved phosphorylation site, can be recognized in this region. The presence of O-phosphoserine at these sites could not be demonstrated, however.     

Nucleotide sequence and expression of a maize H1 histone cDNA.

The first complete amino acid sequence of a H1 histone of a monocotyledonous plant was deduced from a cDNA isolated from a maize library. The encoded H1 protein is 245 amino acid-long and shows the classical tripartite organization of this class of histones. The central globular region of 76 residues shows 60% sequence homology with H1 proteins from dicots but only 20% with the animal H1 proteins. However, several of the amino acids considered as being important in the structure of the nucleosome are conserved between this protein and its animal counterparts. The N-terminal region contains an equal number of acidic and basic residues which appears as a general feature of plant H1 proteins. The 124 residue long and highly basic C-terminal region contains a 7-fold repeated element KA/PKXA/PAKA/PK. Southern-blot hybridization showed that the H1 protein is encoded by a small multigene family. Highly homologous H1 gene families were also detected in the genomes of several more or less closely related plant species. The general expression pattern of these genes was not significantly different from that of these genes encoding the core-histones neither during germination nor in the different tissues of adult maize.     

Isolation of the gene for the testis-specific H1 histone variant H1t

H1t is a testis-specific H1 variant found in pachytene spermatocytes and round spermatids of mammals. The H1t gene was isolated from the Sargent-Bonner library of recombinant lambda bacteriophage containing EcoRI fragments of rat liver DNA using a hybridization probe derived from a chicken H1 variant. The rat H1t gene encodes a 207-amino acid protein (ignoring the initiating methionine) that matches perfectly what is known of the sequence and composition of H1t isolated from rat testes. The gene lacks introns and has good matches to all the consensus sequences known to lie upstream from a variety of H1 genes from diverse organisms. It also has the standard downstream palindromic sequence that specifies the 3'-end of most histone messages. Accordingly, the features of the gene or its environs that restrict its expression to a particular phase of spermatogenesis are not yet evident.     

The gene and the primary structure of acidic ribosomal protein A0 from yeast Saccharomyces cerevisiae which shows partial homology to bacterial ribosomal protein L10

Eukaryotic ribosomes contain an acidic ribosomal protein of about 38 kDa which shows immunological cross-reactivity with the 13 kDa-type acidic ribosomal proteins that are related to L7/L12 of bacterial ribosomes. By using a cDNA clone for 38 kDa-type acidic ribosomal protein A0 from the yeast Saccharomyces cerevisiae, we have cloned a genomic DNA encoding A0 and determined the sequence of 1,614 nucleotides including about 500 nucleotides in the 5'-flanking region. The gene lacks introns and possesses two boxes homologous to upstream activation sequences (UASrpg) in the 5'-flanking region. The amino acid sequence of A0 deduced from the nucleotide sequence shows that A0 shares a highly similar carboxyl-terminal region of about 40 amino acids in length with 13 kDa-type acidic ribosomal proteins, including an identical carboxyl-terminal, DDDMGFGLFD. In the amino-terminal region A0 contains an arginine-rich segment which shows a low but distinct similarity to that of bacterial ribosomal protein L10 through which L10 is thought to bind to 23S rRNA. On the other hand, the carboxyl-terminal half of A0 is enriched with hydrophobic amino acid residues including four pairs of phenylalanine residues which are all conserved in a human homologue.     

Identification of regulatory domains in ADP-ribosyltransferase-1 that determine transferase and NAD glycohydrolase activities

Mono-ADP-ribosyltransferases (ART1-7) transfer ADP-ribose from NAD+ to proteins (transferase activity) or water (NAD glycohydrolase activity). The mature proteins contain two domains, an alpha-helical amino terminus and a beta-sheet-rich carboxyl terminus. A basic region in the carboxyl termini is encoded in a separate exon in ART1 and ART5. Structural motifs are conserved among ART molecules. Successive amino- or carboxyl-terminal truncations of ART1, an arginine-specific transferase, identified regions that regulated transferase and NAD glycohydrolase activities. In mouse ART1, amino acids 24-38 (ART-specific extension) were needed to inhibit both activities; amino acids 39-45 (common ART coil) were required for both. Successive truncations of the alpha-helical region reduced transferase and NAD glycohydrolase activities; however, truncation to residue 106 enhanced both. Removal of the carboxyl-terminal basic domain decreased transferase, but enhanced NAD glycohydrolase, activity. Thus, amino- and carboxyl-terminal regions of ART1 are required for transferase activity. The enhanced glycohydrolase activity of the shorter mutants indicates that sequences, which are not part of the NAD binding, core catalytic site, exert structural constraints, modulating substrate specificity and catalytic activity. These functional domains, defined by discrete exons or structural motifs, are found in ART1 and other ARTs, consistent with conservation of structure and function across the ART family.     

Signature amino acids enable the archaeal L7Ae box C/D RNP core protein to recognize and bind the K-loop RNA motif

The archaeal L7Ae and eukaryotic 15.5kD protein homologs are members of the L7Ae/15.5kD protein family that characteristically recognize K-turn motifs found in both archaeal and eukaryotic RNAs. In Archaea, the L7Ae protein uniquely binds the K-loop motif found in box C/D and H/ACA sRNAs, whereas the eukaryotic 15.5kD homolog is unable to recognize this variant K-turn RNA. Comparative sequence and structural analyses, coupled with amino acid replacement experiments, have demonstrated that five amino acids enable the archaeal L7Ae core protein to recognize and bind the K-loop motif. These signature residues are highly conserved in the archaeal L7Ae and eukaryotic 15.5kD homologs, but differ between the two domains of life. Interestingly, loss of K-loop binding by archaeal L7Ae does not disrupt C′/D′ RNP formation or RNA-guided nucleotide modification. L7Ae is still incorporated into the C′/D′ RNP despite its inability to bind the K-loop, thus indicating the importance of protein–protein interactions for RNP assembly and function. Finally, these five signature amino acids are distinct for each of the L7Ae/L30 family members, suggesting an evolutionary continuum of these RNA-binding proteins for recognition of the various K-turn motifs contained in their cognate RNAs.     

Histone H1 variants differentially inhibit DNA replication through an affinity for chromatin mediated by their carboxyl-terminal domains

Multiple forms of histone H1 are found in most mammalian tissues, and diversity in their temporal and spatial expression likely corresponds to diversity in function. Here, using Xenopus egg extracts, we show that while the somatic H1s significantly inhibit DNA replication in Xenopus sperm nuclei, little or no inhibition is seen in the case of the testes-specific variant, H1t. We suggest that differences in H1-chromatin interactions might explain some of the diversity in H1 function. To demonstrate this, we show that the somatic H1 variants preferentially assemble into chromatin relative to H1t. Differences in chromatin structure are seen depending on whether chromatin assembly occurs in the presence of somatic H1s or H1t. These data suggest that the mechanistic basis for some of the functional differences of H1 variants lies in their relative affinity for chromatin. Using a series of domain-switch mutants of H1(0) and H1t we identify the H1 carboxyl-terminal domains as the domains responsible for the differential affinity for chromatin and, concurrently, for the differential effects of H1 variants upon DNA replication.     

Polyadenylated H3 histone transcripts and H3 histone variants in alfalfa

Histone H3 mRNAs were found in polyA(+) fractions of total RNA prepared from alfalfa plants, calli and somatic embryos. The sequence analysis of cDNAs revealed the presence of a polyA tail on independent alfalfa H3 mRNAs. A highly conserved sequence motif AAUGAAA identified about 20bp upstream from the 3' ends of the alfalfa H3 cDNAs was suggested to be one of the possible regulatory elements in the 3' end formation and polyadenylation. Three out of the four analysed H3 cDNAs have more than 97% homology with a genomic clone and encode the same protein. While the fourth represents a minor species with only 78.8% homology to the coding region of the genomic clone and encodes a H3 histone with four amino acid replacements. On the basis of compilation analysis we suggest a consensus sequence for plant H3 histones which differs from that of animal's by four amino acid changes.