The distribution of Tap2 alleles among laboratory rat RT1 haplotypes

We are reporting the cDNA sequences of Tap2 from two cim ^a and two cim ^b rat strains. Comparison of the cDNA sequences shows that these alleles fall into two groups, which we refer to as Tap2-A and Tap2-B. We found that alleles from the Tap2-B group are more closely related to the mouse homologue than are Tap2-A alleles, and among the 48 nucleotides which differ between the Tap2-A and Tap2-B cDNAs, three affect restriction sites. We defined pairs of oligonucleotides which allow amplification of the regions bearing these restriction sites from genomic DNA or cDNA, and this technique has been successful for the genotyping of all of the 56 laboratory strains of Rattus norvegicus tested and for five cell lines tested so far. All 14 known RT1 standard haplotypes were tested, and 7 found to belong to the Tap2-B group, and 7 to Tap2-A. We also found that intron sizes among the alleles of the Tap2-B group fall into two subgroups, providing further insight into the phylogency of these various haplotypes.     

Sequences from the beginning of the fiber messenger RNA of adenovirus-2.

Small restriction fragments, from around co-ordinate 86.6 on the adenovirus-2 genome, have been used as primers for direct DNA sequence analysis by Sanger's (Sanger et al., 1977) chain termination method with Ad2² DNA as template. The genomic sequences obtained have been compared with sequences deduced using fiber messenger RNA from Ad2 or Ad2⁺ ND5-infected cells as template. With one primer, Hha 54, the sequences complementary to mRNA match those of the genome for 10 nucleotides but then differ from those found on the genome because this primer hybridizes near the point at which the leader sequence becomes joined to the main body of fiber mRNA. Using Ad2⁺ ND5 fiber mRNA as template, the sequence beyond the point of divergence matches the known sequence of the third leader component of one of the late Ad2 mRNAs, that encoding the hexon polypeptide. With Ad2 fiber mRNA, a heterogeneous sequence continuation is found, in accordance with earlier findings that two major species of fiber mRNA are present, which differ in the nature of the leader component joined to the main body of fiber mRNA (Chow &; Broker, 1978). Nevertheless, the data suggest that both leader components appear to become joined to the same nucleotide at the start of the main body of fiber mRNA.The AUG codon, which probably encodes the N-terminus of the fiber polypeptide, occurs just two nucleotides beyond the point at which these leader segments become spliced to the main body of fiber mRNA.     

Vitellogenin in Xenopus laevis is encoded in a small family of genes.

Vitellogenin, the yolk protein precursor, is produced in X. laevis liver from a 6.3 kilobase (kb) mRNA. Sequences of this mRNA have been transcribed into cDNA and cloned in E. coli. Some properties of 21 of these cloned DNAs, ranging in size from 1 to 3.7 kb, have been reported by Wahli et al. (1978b). This paper reports restriction endonuclease mapping, cross hybridization, heteroduplex mapping in the electron microscope and heteroduplex melting experiments with these DNAs. We conclude that the cloned DNAs fall into two main groups of sequences which differ from each other in approximately 20% of their nucleotides. Each main group contains two subgroups which differ from each other by about 5% sequence divergence. By hybridizing cloned DNAs with restricted genomic DNA, we showed that sequences corresponding to all four sequence groups are present in a single animal. Furthermore, we have obtained tentative evidence for the presence of large intervening sequences in genomic vitellogenin DNA. Analysis of R loop molecules demonstrated that all four sequences are present in the vitellogenin mRNA population purified from individual animals. While some alternate explanations are not entirely excluded, we suggest that vitellogenin is encoded by a small family of related genes in Xenopus.     

Physical Map of the Channel Catfish Virus Genome: Location of Sites for Restriction Endonucleases EcoRI, HindIII, HpaI, and XbaI

The overall arrangement of nucleotide sequences in the DNA of channel catfish virus has been studied by cleavage with four restriction endonucleases. Physical maps have been developed for the location of sites for EcoRI, HindIII, HpaI, and XbaI. The sum of the molecular weights of fragments generated by each restriction enzyme indicates a molecular weight of approximately 86 × 10⁶ for the channel catfish virus genome. Fragments corresponding to the molecular ends of channel catfish virus DNA have been identified by their sensitivity to exonuclease treatment. The distribution of restriction sites in the genome shows that sequences included in a 12 × 10⁶-molecular weight region at one end are repeated with direct polarity at the other end, and that the overall genomic sequence order is nonpermuted.     

Organization of human δ- and β-globin genes in cellular DNA and the presence of intragenic inserts

We have analyzed human cellular DNA for its δ- and β-globin gene sequence content by separation of restriction enzyme fragments by agarose gel electrophoresis; transfer of the DNA fragments to nitrocellulose filters; hybridization of filters with ³²P-β-globin cDNA; and analysis by autoradiography. A short cDNA has been used to identify specifically the 3′ end of the genes and to orient the fragments. A comparison of the globin gene fragments generated by normal and Lepore DNA has been used to distinguish fragments representing DNA sequences between the δ and β genes and those containing sequences flanking either 5′ to the δ gene or 3′ to the β gene. The results indicate that unique restriction fragments are presented in normal DNA and absent in Lepore DNA, and allow preliminary ordering of these fragments on a restriction enzyme map. In addition, the Lepore, δ- and β-globin genes have been found to contain at least one inserted nucleotide sequence of about 1000 bases which is not represented in mature globin mRNA.     

Isolation of two cDNA clones coding for larval hemolymph proteins of Galleria mellonella

Galleria mellonella a group of four larval hemolymph proteins (LHP) (74, 76, 81 and 82 kDa), which had been earlier shown to be storage proteins, exhibit a stage-specific synthetic pattern. The 82 kDa LHP is synthesized only in day-3 to day-5 last instar larvae, while the other three LHPs are synthesized both in the penultimate (six) and the last instar larvae. None of these LHPs are synthesized in day-0 last instar. With a view to isolate one or more cDNA clones corresponding to these LHPs a cDNA library was prepared in pBR322 starting with poly(A)⁺ RNA from day-5 last instar larval fat body. By differential screening of 714 clones with poly(A)⁺ RNA 39 day-5 larval stage-specific clones were isolated. Two of these clones, designated as 26–38 and 17–36, had 1200–1300 base pair cDNA inserts. Their cDNA inserts did cross hybridize to each other, exhibited different restriction endonuclease digestion patterns and hybridized in northern blots to transcrips of different sizes, thereby suggesting that they represent two separate genes. In addition, the genomic fragments that hybridized in southern blots to the two cDNAs differed in their size. On translation, mRNAs hybrid selected by 26–38 and 17–36 cDNAs produced 76 and 79 kDa polypeptides respectively. Both these genes are expressed in the fat body but not in the midgut, silk glands, Malpighian tubules or carcass. While 26–38 was expressed both in the sixth and seventh (last) instars, 17–36 was expressed only in the last instar. On the basis of tissue and developmental stage specificity of their expression and the sizes of their hybrid selected translation products, these clones are tentatively identified as two LHP-specific cDNA clones. The genes coding for these LHPs appear to be single copy genes.     

Phylogenetic Analysis of Anaerobic Psychrophilic Enrichment Cultures Obtained from a Greenland Glacier Ice Core

The examination of microorganisms in glacial ice cores allows the phylogenetic relationships of organisms frozen for thousands of years to be compared with those of current isolates. We developed a method for aseptically sampling a sediment-containing portion of a Greenland ice core that had remained at −9°C for over 100,000 years. Epifluorescence microscopy and flow cytometry results showed that the ice sample contained over 6 × 10⁷ cells/ml. Anaerobic enrichment cultures inoculated with melted ice were grown and maintained at −2°C. Genomic DNA extracted from these enrichments was used for the PCR amplification of 16S rRNA genes with bacterial and archaeal primers and the preparation of clone libraries. Approximately 60 bacterial inserts were screened by restriction endonuclease analysis and grouped into 27 unique restriction fragment length polymorphism types, and 24 representative sequences were compared phylogenetically. Diverse sequences representing major phylogenetic groups including alpha, beta, and gamma Proteobacteria as well as relatives of the Thermus, Bacteroides, Eubacterium, and Clostridium groups were found. Sixteen clone sequences were closely related to those from known organisms, with four possibly representing new species. Seven sequences may reflect new genera and were most closely related to sequences obtained only by PCR amplification. One sequence was over 12% distant from its closest relative and may represent a novel order or family. These results show that phylogenetically diverse microorganisms have remained viable within the Greenland ice core for at least 100,000 years.     

Evolution of the chordate muscle actin gene

Summary The ascidians Styela plicata, S. clava, and Mogula citrina are urochordates. The larvae of urochordates are considered to morphologically resemble the ancestral vertebrate. We asked whether larval and adult ascidian muscle actin sequences are nonmusclelike as in lower invertebrates, musclelike as in vertebrates, or possess characteristics of both. Nonmuscle and muscle actin cDNA clones from S. plicata were sequenced. Based on 27 diagnostic amino acids, which distinguish vertebrate muscle actin from other actins, we found that the deduced protein sequences of ascidian muscle actins exhibit similarities to both invertebrate and vertebrate muscle actins. A comparison to muscle actins from different vertebrate and invertebrate phylogenetic groups suggested that the urochordate muscle actins represent a transition from a nonmusclelike sequence to a vertebrate musclelike sequence. The ascidian adult muscle actin is more similar to skeletal actin and the larval muscle actin is more similar to cardiac actin, which indicates that the divergence of the skeletal and cardiac isoforms occurred before the emergence of urochordates. The muscle actin gene may be a powerful probe for investigating the chordate lineage. Offprint requests to: C.R. Tomlinson     

Extrachromosomal ribosomal RNA genes in Tetrahymena: structure and evolution

The macronuclear ribosomal RNA genes from a number of strains within several species of Tetrahymena have been characterized. Restriction enzyme analysis revealed that individual strains all contained entirely homogeneous populations of extrachromosomal palindromic ribosomal DNA, varying in molecular size from 12 × 10⁶ to 14 × 10⁶ in different strains. Considering that the evolutionary distance among some of the species is estimated to be of the order of 10⁶ years, the rDNA from all the species exhibited a strikingly high similarity in the localization of their restriction sites. Nevertheless, differences both inside and outside the gene region were clearly detectable, showing that the rDNA sequences have diverged in all species.Genetic polymorphism with respect to rDNA structure exists in Tetrahymena, but seems to be rare. In only two out of five species examined (T. borealis and T. pigmentosa) interbreeding strains differing in rDNA structure were found. While the differences detected in the T. borealis rDNA were confined to a small size difference located at the non-coding ends of the molecule, several differences were detected in the rDNA from the T. pigmentosa strains. One of the differences was shown to be due to the presence of an intervening sequence within the structural gene for 26 S rRNA in some of the strains. An intervening sequence of similar size located at the same position within the 26 S gene region was found by R-loop mapping in all strains of the species T. thermophila. Restriction enzyme analysis indicates that the rDNA from two other species contains a similar intervening sequence, and we therefore suggest that the size and localization of the intervening sequence is evolutionarily stable. The two intervening sequences examined so far, however, are not identical, as revealed by restriction enzyme mapping.     

The nucleotide sequence recognized by the Escherichia coli K12 restriction and modification enzymes.

The sites recognized by the Escherichia coli K12 restriction endonuclease were localized to defined regions on the genomes of phage φXsK1, φXsK2, and G4 by the marker rescue technique. Methyl groups placed on the genome of plasmid pBR322 by the E. coli K12 modification methylase were mapped in HinfI fragments 1 and 3, and HaeIII fragments 1 and 3. A homology of seven nucleotides in the configuration: 5′-A-A-C .. 6N .. G-T-G-C-3′, where 6N represents six unspecified nucleotides, was found among the DNA sequences containing the five EcoK sites of φXsK1, φXsK2, G4, and pBR322. Three lines of evidence indicate that this sequence constitutes the recognition site of the E. coli K12 restriction enzyme. The C in 5′-A-A-C and the T in 5′-G-T-G-C are locations of mutations leading to loss or gain of the site and thus are positions recognized by the enzyme. This sequence does not occur on φXam3cs70, simian virus 40 (SV40), and fd DNAs which do not possess EcoK sites, and occurs only once on φXsK1, φXsK2, and G4 DNAs, and twice on pBR322 DNA. In order to prove that all seven conserved nucleotides are essential for the recognition by the E. coli K12 restriction enzyme, the nucleotide sequences of φX174, G4, SV40, fd, and pBR322 were searched for sequences differing from the sequence 5′-A-A-C .. 6N .. G-TG-C-3′ at only one of the specified positions. It was found that sequences differing at each of the specified positions occur on DNA sequences that do not contain the EcoK sites. Thus, the recognition site of the E. coli K12 restriction enzyme has the same basic structure as that of the EcoB site (Lautenberger et al., 1978). In each case there are two domains, one containing three and the other four specific nucleotides, separated by a sequence of unspecified bases. However, the unspecified sequence in the EcoK site must be precisely six bases instead of the eight found in the EcoB site. Alignment of the EcoK and EcoB sites suggests that four of the seven specified nucleotides are conserved between the sequences recognized by these two allelic restriction and modification systems.     

Sequence and sequence variation within the 1.688 g/cm3 satellite DNA of Drosophila melanogaster.

We have determined the complete nucleotide sequence of the monomer repeating unit of the 1.688 g/cm³ satellite DNA from Drosophila melanogaster. This satellite DNA, which makes up 4% of the Drosophila genome and is located primarily on the sex chromosomes, has a repeat unit 359 base-pairs in length. This complex sequence is unrelated to the other three major satellite DNAs present in this species, each of which contains a very short repeated sequence only 5 to 10 base-pairs long. The repeated sequence is more similar to the complex repeating units found in satellites of mammalian origin in that it contains runs of adenylate and thymidylate residues. We have determined the nature of the sequence variations in this DNA by restriction nuclease cleavage and by direct sequence determination of (1) individual monomer units cloned in hybrid plasmids, (2) mixtures of adjacent monomers from a cloned segment of this satellite DNA, (3) mixtures of monomer units isolated by restriction nuclease cleavage of total 1.688 g/cm³ satellite DNA. Both direct sequence determination and restriction nuclease cleavage indicate that certain positions in the repeat can be highly variable with up to 50% of certain restriction sites having altered recognition sequences. Despite the high degree of variation at certain sites, most positions in the sequence are highly conserved. Sequence analysis of a mixture of 15 adjacent monomer units detected only nine variable positions out of 359 base-pairs. Total satellite DNA showed only four additional positions. While some variability would have been missed due to the sequencing methods used, we conclude that the variation from one repeat to the next is not random and that most of the satellite repeat is conserved. This conservation may reflect functional aspects of the repeated DNA, since we have shown earlier that part of this sequence serves as a binding site for a sequence-specific DNA binding protein isolated from Drosophila embryos (Hsieh &; Brutlag, 1979).     

Cloning and expression of apolipophorin-III from the common cutworm,Spodoptera litura

We have cloned apolipophorin-III (apoLp-III) cDNA from adult fat body of Spodoptera litura. The sequence encodes a 188 amino acid polypeptide including a 22 amino acid leader peptide. The circular dichroism spectrum from the purified apoLp-III indicated a considerable content of α-helix. Sequence alignment showed that S. Litura apoLp-III has a relatively high degree of sequence identity with the apoLps-III of lepidopteran, Manduca sexta (72%), Galleria mellonella (67%), Bombyx mori (60%). These alignments with four lepidopteran apoLps-III showed highly identical residues and conservative replacements at a degree of 86%. Levels of mRNA from last instar larval fat body and adult fat body were compared through Northern blot analysis using ³²P-labeled 704 bp apoLp-III cDNA probe. A 850 bp mRNA was detected in both stages and mRNA level of day 1 adult fat body was much higher than that of last instar larval fat body. The tissue-distribution of apoLp-III mRNA in adult ovary and testis was also examined and we confirmed the presence of apoLp-III mRNA in ovary and testis although apoLp-III was expressed in these tissues at very low levels compared with the adult fat body. Arch. Insect Biochem. Physiol. 39:166–173, 1998. © 1998 Wiley-Liss, Inc.     

Cloning of large-conductance Ca-activated K channel α-subunits in mouse cardiomyocytes

Large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels are widely distributed in cellular membranes of various tissues, but have not previously been found in cardiomyocytes. In this study, we cloned a gene encoding the mouse cardiac BK_Ca channel α-subunit (mCardBKa). Sequence analysis of the cDNA revealed an open reading frame encoding 1154 amino acids. Another cDNA variant, identical in amino acid sequence, was also identified by sequence analysis. The nucleotide sequences of the two mCardBKa cDNAs, type 1 (mCardBKa1) and type 2 (mCardBKa2), differed by three nucleotide insertions and one nucleotide substitution in the N-terminal sequence. The amino acid sequence demonstrated that mCardBKa was a unique BK_Ca channel α-subunit in mouse cardiomyocytes, with amino acids 41-1153 being identical to calcium-activated potassium channel SLO1 and amino acids 1-40 corresponding to BK_Ca channel subfamily M alpha member 1. These findings suggest that a unique BK_Ca channel α-subunit is expressed in mouse cardiomyocytes.