Germ line-restricted supernumerary (B) chromosomes in Eptatretus okinoseanus.

Cytogenetic studies were performed on two types of Japanese hagfish (Eptatretus okinoseanus) that eliminate about 45% (type A) and 55% (type B) of their DNA from presumptive somatic cells during the differentiation of somatic cells. The observations revealed inter- and intraindividual variations in the number of chromosomes in germ cells of both types of hagfishes. Although the modal number of chromosomes in the germ cells was 54 in both types, the percentage of cells with the modal number was rather low (38.6% [51/132] in five specimens of type A and 22.7% [25/110] in eight specimens of type B). In addition, one of seven type B specimens clearly had a modal number of 62 chromosomes. Another specimen of type B had a bimodal distribution of chromosome numbers, with peaks of 54 and 59 chromosomes. The observation of interindividual variations was supported by data on the amount of DNA in germ cells of type B specimens. However, these variations were rarely observed in somatic cells. These results suggest that supernumerary (B) chromosomes are maintained in germ cells and are eliminated together with some other chromosomes and/or chromatin from somatic cells.     

Chromosome elimination in the Japanese hagfish, Eptatretus burgeri (Agnatha, Cyclostomata)

The modal number of chromosomes in Eptatretus burgeri was 36 in 297 (81.8%) of 363 somatic cells, 52 in 13 (30.2%) of 43 spermatogonia, and 25 (46.8%) or 26 (40.3%) in 162 of 186 first spermatocytes. The relative amount of DNA in a somatic cell to that in a spermatogonium averaged 79.2%. C-band-positive chromatin was observed along almost the entire length of the associated dumbbell-shaped bivalents and part of two other bivalents in the metaphases of first spermatocytes, but was rarely observed in somatic cells. These results indicate that, in E. burgeri, chromosome elimination takes place during the early stages of cleavage, except for the ancestral germ-line cells.     

Nucleotide sequence of a highly repeated DNA sequence and its chromosomal localization in Allium fistulosum

A highly repeated DNA sequence with a repeating unit of approximately 380bp was found in EcoRV digests of the total genomic DNA of Allium fistulosum. Three independent clones containing this unit were isolated, and their repeating units sequenced. These units showed more than 94% sequence homology, and the copy number was estimated to be about 2.8×10⁶ per haploid genome. In situ hybridization, with the repeating unit as a probe, and C-banding analyses indicated that the repeated DNA sequence of A. fistulosum is closely associated with the major C-heterochromatin in the terminal regions of all 16 chromosomes at mitotic metaphase. The characters of the repeating unit are similar to those of the A. cepa unit, which is taxonomically closely related to A. fistulosum.     

Heterochromatin and highly repeated DNA sequences in rye (Secale cereale)

Secale cereale DNA, of mean fragment length 500 bp, was fractionated by hydroxylapatite chromatography to allow recovery of a very rapidly renaturing fraction (C₀t 0–0.02). This DNA fraction was shown to contain several families of highly repeated sequence DNA. Two highly repeated families were purified; (1) a fraction which renatured to a density of 1.701 g/ cc and comprised 2–4% of the total genome, and (2) polypyrimidine tract DNA which comprised 0.1% of the total genome. The 1.701 g/cc DNA consisted of short sequence repeat units (5–50 bp long) tandemly repeated in blocks 30 kb long, while a portion of the polypyrimidine tract DNA behaved as part of a much larger block of tandemly repeated sequences. The chromosomal location of these sequences was determined by the in situ hybridisation of radioactive, complementary RNA to root tip mitotic chromosomes and showed the 1.701 g/cc sequences to be largely limited to the telomeric blocks of heterochromatin, accounting for 25–50% of the DNA present in these parts of the chromosomes. The polypyrimidine tracts were distributed at interstitial locations with 20–30% of the sequences at three well defined sites. The combined distributions of the 1.701 g/cc DNA sequences and polypyrimidine tracts effectively individualised each rye chromosome thus providing a sensitive means of identifying these chromosomes. The B chromosomes present in Secale cereale cv. Unevita, did not show defined locations for the sequences analysed. — The data are discussed in terms of the structure of the rye genome and the generality of the observed genomic arrangement of highly repeated sequence DNA.     

Evolutionary implications of lactate dehydrogenases (LDHs) of hagfishes compared to lampreys: LDH cDNA sequences from Eptatretus burgeri, Paramyxine atami and Eptatretus okinoseanus

Heart muscles of hagfishes Paramyxine atami and Eptatretus okinoseanus express the B4 isozyme of lactate dehydrogenase [L-LDH: NAD oxidoreductase, EC 1.1.1.27] (LDH-B4) whereas their skeletal muscles express LDH-A4. To examine the relationship of hagfish LDHs to lamprey and other vertebrate LDHs, we determined the cDNA sequences of LDH-A from three hagfishes and compared them with previously published sequences. A phylogenic tree shows that hagfishes diverged just after lampreys. The deduced amino acid sequences showed ten regions common to all vertebrate LDHs examined, i.e., the active site, the pocket recognizing the substrate-coenzyme complex, part of a loop at the surface, and the substrate binding site. The cyclostomate-specific regions (S1, S2) were located in the neighborhood of the active site loop. Three regions, IGS1, IGS2 and IGS3, seem to have altered their structures during the differentiation of LDH isozymes, and the regions remain in LDH-B of vertebrates hitherto examined. IGS2 and IGS3, which are in the neighborhood of the active site, may regulate catalytic activity. There were differences in six amino acid residues (6, 10, 20, 156, 269, and 341) in LDHs of hagfishes. These differences might reflect the tolerance to high pressure and low temperature of LDHs from hagfishes at different habitat depths.     

Highly repetitive DNA families restricted to germ cells in a Japanese hagfish (Eptatretus burgeri): a hierarchical and mosaic structure in eliminated chromosomes

It is known that in eight hagfishes chromosome elimination occurs during early embryogenesis. The eliminated chromosomes are mostly C-band positive, so that none of the somatic cells have any C-band-positive chromatin. Recently, some highly repetitive DNA sequences have been reported as eliminated elements in these hagfishes based on molecular biological methods. However, no germline-restricted repetitive DNA have been directly isolated from the Japanese hagfish Eptatretus burgeri, from which approximately 21% of the total DNA is eliminated from presumptive somatic cells. Through electrophoretic investigation after digestion with restriction endonucleases, two DNA families that are restricted to germline DNA were isolated. Molecular cloning and sequence analysis revealed that these families are composed of closely related sequences of 64 and 57bp in length, respectively. Southern blot hybridization revealed that the two DNA families are restricted to germline DNA and were thus named EEEb1 and EEEb2, respectively. Moreover, these eliminated elements were highly and tandemly repeated, and it is predicted that they might amplify by saltatory replication and have evolved in a concerted manner. By densitometric scanning, EEEb1 and EEEb2 were found to amount to make up approximately 18.5 and 0.024% of the total germline genomic DNA, accounting for 88.6% of the total eliminated DNA. A fluorescence in situ hybridization experiment demonstrated that EEEb1 is located on all C-band-positive chromosomes that are limited to germ cells, suggesting that EEEb1 is the primary component of eliminated DNA of E. burgeri.     

Acrosome reaction in spermatozoa of the hagfish Eptatretus burgeri (Agnatha)

Fertilization of the hagfish or myxiniformes, a member of the most primitive vertebrate group and an animal of phylogenic interest, is unknown. Here, induction of an acrosome reaction for spermatozoa in the hagfish, Eptatretus burgeri, was successfully achieved by treatment of mature spermatozoa with ionomycin and excess Ca2+. The spermatozoon produced an acrosomal process that elongated from the apex of the long sperm head. The reaction bears resemblance to that of invertebrate spermatozoa rather than that of vertebrate spermatozoa. The result provides insights into the phylogenetical changes that have occurred in this sperm reaction.     

Immunocytochemical localization of somatostatin and vasotocin in the brain of the Pacific hagfish,Eptatretus stouti

Summary The brain of the Pacific hagfish, Eptatretus stouti, was studied immunocytochemically using antisera against somatostatin (SRIH), arginine vasopressin (AVP), and adrenocorticotropic hormone (ACTH). SRIH-immunoreactive perikarya were distributed bilaterally in the postoptic nucleus and in the hypothalamic nucleus. Although several short, stained fibers were observed in the vicinity of the perikarya, SRIH-immunoreactivity was not found in the neurohypophysis, nor in other parts of the brain. On the other hand, presumed arginine vasotocin (AVT) perikarya were distributed in an arc-shaped region extending from the posterior part of the preoptic nucleus to the anterior-most end of the hypothalamic nucleus and projected their fibers to the neurohypophysis. Most presumptive AVT perikarya were located close to the paired prehypophysial arteries near the anterior end of the postoptic nucleus. In the neurohypophysis, abundant presumptive AVT-fibers terminated in the posterior dorsal wall, although some fibers terminated in the anterior dorsal wall and only a few fiber endings were found in the ventral wall. No ACTH-positive cells were detected in the hagfish brain or in the pituitary gland.Supported from a grant from the National Science Foundation PCM 8141393     

Complete mitochondrial DNA of the hagfish, Eptatretus burgeri: the comparative analysis of mitochondrial DNA sequences strongly supports the cyclostome monophyly

The phylogenetic position of cyclostomes, i.e., the relationships between hagfishes, lampreys, and jawed vertebrates is an unresolved problem. Anatomical data support the paraphyly of cyclostomes, whereas nuclear genes data support monophyly of cyclostomes. Previous results obtained using mitochondrial DNA are ambiguous, presumably due to a lack of informative sequences. By adding the complete mtDNA of a hagfish, Eptatretus burgeri, we have generated a novel data set for sequences of hagfishes and of lampreys. The addition of this mtDNA sequence to the 12 taxa we have already used becomes sufficient to obtain unambiguous results. This data set, which includes sequences of mtDNA of animals closely related to the lamprey/hagfish node, was used in a phylogenetic analysis with two independent statistical approaches and unequivocally supported the monophyly of cyclostomes. Thus molecular data, i.e., our results and those obtained using nuclear genes, conclude that hagfishes and lampreys form a clade.     

FMRFamide-like immunoreactivity in the brain of the Pacific hagfish,Eptatretus stouti (Myxinoidea)

Summary The distribution of FMRFamide-like immunoreactivity was investigated in the brain of a myxinoid, the Pacific hagfish,Eptatretus stouti, by means of immunocytochemistry. In the forebrain, labelled cell bodies occurred in the infundibular nucleus of the hypothalamus and some closely adjacent nuclei. Labelled fibers formed a diffuse network in the forebrain, but there was no evidence for the presence of intracerebral ganglionic cells of the terminal nerve or a central projection of the terminal nerve. In the hindbrain, a group of labelled cells was found in the trigeminal sensory nucleus. A distinet terminal arborization occurred in the ventrally adjacent nucleus A of Kusunoki and around the nuclei of the branchial motor column. These findings suggest that FMRFamide may play a role in the central control of branchiomotor activity.     

Identification and chromosomal location of tandemly repeated DNA sequences in Allium cepa

A 314-bp tandemly repeated DNA sequence, named pAc074, was characterized in Allium cepa by fluorescence in situ hybridization (FISH) analyses using random amplified fragment as probe. The nucleotide sequences of the clone pAc074 is partially homologous to the satellite DNA sequences, ACSAT1, ACSAT2, and ACSAT3, of A. cepa with 81%, 81% and 78% similarity, respectively. Our sequential C-banding and FISH with pAc074 probe also clearly showed a close relation between Cheterochromatin at telomeric region and pAc074 sequences on all the chromosomes except on chromosome 6. On the long arm of chromosome 7, pAc074 sequences appeared as interstitial band which did not correspond to C-heterochromatin bands. Instead, the C-heterochromatin bands corresponded with the 5S rDNA signals. This is the first evidence of simultaneous banding of the 5S rDNA and C-band in A. cepa.     

Metabolic-morphologic events in the integument of the Pacific hagfish (Eptatretus stoutii)

Summary Light- and electron-microscopic autoradiography were used to obtain a coordinated metabolic-morphologic view of some of the events of cellular differentiation that occur across the epidermis of the Pacific hagfish (Eptatretus stoutii) and which enable this animal to secrete copious amounts of mucus. As judged by epidermal incorporation of [³H]-thymidine in vivo, about 98 % of DNA replication is confined to the basal three layers of the total of 6–8 layers of cells. Small mucous cells (SMC), the most numerous of the three major cell types involved in mucigenesis, show in vitro and in vivo radioincorporation profiles of [³H]-L-lysine and [³H]-D-glucosamine which differ markedly from those of [³H]-L-fucose and [³H]-D-galactose. Time-course incorporation profiles (mean silver grains/cell and percentage of cells with at least one cluster of silver grains) of [³H]-L-lysine and [³H]-D-glucosamine not only reflected the metabolic activities of cell renewal and differentiation in basally-located cells but also the high mucigenic activity in cells near the epidermal surface. By contrast, [³H]-L-fucose and [³H]-D-galactose were mainly incorporated by the more mature SMC in juxtanuclear regions near Golgi complexes and newly formed secretory vesicles. The intensity of [³H]-fucose labeling appeared proportional to the intensity of histochemical staining of the apical cytoplasm. The prominent capsule, within SMC in basal and lateral regions, which arises from a tight intermingling of tonofilaments, appears to restrict secretory vesicles to apical regions while the cell progressively differentiates and migrates to the epidermal surface. The other mucigenic cell types, large mucous cells and thread cells, each show distinctive differentiation and radioincorporation patterns.This study was supported in part by General Research Support Grant, FR5366 of the NIH, Minnesota Medical Foundation Grants, and the Asthmatic Children's Aid of Chicago, Illinois. The authors wish to thank Karen Brintzenhofe and Doreen Fleetwood for their assistance     

Cytological localization of inverted repeated DNA sequences in Vicia faba

Inverted repeated DNA sequences have been isolated from sheared Vicia faba DNA by hydroxylapatite column chromatography, treated with nuclease S₁, tritiated by the nick translation method and hybridized in situ on squashes of Vicia faba root tips. Silver grains appear grouped in a rather limited portion of interphase nuclei and form a sort of band across them. The central regions of metaphase chromosomes are preferentially labeled, labeling being excluded from telomeres, centromeres and secondary constrictions. These results are briefly discussed in relation to those obtained in other species and the functional significance of inverted repeats.     

Highly repetitive DNA sequences that are restricted to the germ line in the hagfish Eptatretus cirrhatus: a mosaic of eliminated elements

The New Zealand hagfish, Eptatretus cirrhatus, is known to eliminate parts of its chromosomes during embryogenesis from presumptive somatic cells. Electrophoresis of germ line and somatic DNAs of this species, after treatment with the restriction endonucleases DraI and EcoRI, revealed three fragments of DNA that were restricted to the germ line. DNA filter hybridization experiments demonstrated that these fragments were present almost exclusively in the germ line DNA of E. cirrhatus and that they were highly and tandemly repeated. Thus, these DNA fragments appeared to be eliminated during embryogenesis. Moreover, one fragment (a DraI fragment) cross-hybridized with the germ line DNA from other species of hagfish, namely, Eptatretus okinoseanus and Paramyxine atami. Molecular cloning and sequence analysis revealed that the DraI fragment was composed mainly of closely related sequences of 85 bp in length and that this sequence was about 75% homologous to the sequence of EEEo2 (eliminated element of E. okinoseanus 2) which is a germ line-restricted and highly repetitive sequence that was isolated previously from E. okinoseanus. The other two fragments were composed of three families of closely related sequences that were 172 bp long (designated EEEc1), 61 bp long (EEEc2) and 54 bp long (EEEc3). Fluorescence in situ hybridization experiments revealed that each eliminated element was distributed on several chromosomes that are limited to germ cells. EEEo2 was dispersed on 12 C-band-positive chromosomes. EEEc1 and EEEc3 were dispersed on all C-band-positive and several C-band-negative chromosomes. By contrast, EEEc2 was located to terminal regions of several C-band-negative chromosomes. These results suggest that the eliminated chromosomes in hagfish are mosaics of highly repeated, germ line-restricted families of DNA sequences. Received: ██; in revised form: 25 October 1997 / Accepted: ██     

FMRF -amide-like immunoreactivity in brain and pituitary of the hagfish Eptatretus burgeri (Cyclostomata)

Summary Paraffin sections of brain and pituitary of the hagfish Eptatretus burgeri were immunostained with an antiserum to FMRF-amide. Immunoreactivity was visible in a large number of neurons in the posterior part of the ventromedial hypothalamus and in long neuronal processes extending cranially from the hypothalamus to the olfactory system and caudally to the medulla oblongata. FMRF-amide-like immunoreactivity was also found in cells of the adenohypophysis. These observations suggest that the hagfish possesses a brain FMRF-amide-like transmitter system and pituitary cells containing FMRF-amide-like material.Antisera to ACTH, -MSH and pancreatic polypeptide gave no immunoreaction in hagfish brain or pituitary. D aspartic acid - F phenylalanine - L leucine - M methionine - R arginine; - W tryptophan - Y tyrosine     

Organization of DNA sequences highly repeated in tandem in rice genomes

Digestion of the total genomic DNA from rice Oryza sativa L. cv. C5924 with EcoRI generated an intense band of a DNA fragment of about 0.36 kb long. The DNA fragment cloned into pUC19 was used to hybridize with the total rice genomic DNA partially digested with EcoRI. A ladder of bands of DNA fragments with multiplied length of 0.36 kb was observed, demonstrating that this sequence occurs in tandem in the genome. The copy number of the sequence estimated by dot blot hybridization analysis was 2000-3000 copies per haploid genome from callus or seedling of C5924. This sequence was present in other O. sativa cultivars, such as Sasanishiki in 700-900 copies, Koshihikari in 3400-4300, and Nipponbare in 4600-6000 copies. Another rice species, O. glaberrima, also had this sequence in 540-680 copies, but four lines of foxtail millet had none. The DNA fragments containing the repeated sequences in Nipponbare were then cloned into lambda EMBL3, and sequences of nine units consecutively repeated and an AT-rich sequence connected with them in a phage clone could be determined. Each repeating unit showed sequence divergency mostly by substitution of bases in a range from 3% to 7%, when compared with a 355-bp consensus sequence. Analyses of the substituted bases indicate that these are due to spontaneous mutations which occurred at random, after reiteration of a unit sequence by unequal crossing over events. Gene conversion within the repeated sequences might have further diversified their sequences.     

cDNA cloning of a mannose-binding lectin-associated serine protease (MASP) gene from hagfish (Eptatretus burgeri)

Hagfish, agnathan cyclostome, is the most primitive extant vertebrate and its complement (C) system seems to be a primordial system in comparison with a well-developed C system in gnathostome vertebrates. From a phylogenic perspective of defense mechanisms, we have isolated complement C3 from the serum of hagfish (Eptatretus burgeri). In this study, we first attempted to identify a hagfish Bf or C2 as a C3 convertase by RT-PCR using degenerative primers designed on the basis of the conserved amino acid stretches among the several kinds of serine proteases. Contrary to our expectation, homology search of cloned RT-PCR product suggested that there was a partial cDNA encoding the homologue of neither Bf nor C2 but a mannose-binding lectin-associated serine protease (MASP). Analyses of a full-length cDNA clone isolated from a hagfish liver cDNA library by using the partial cDNA as a probe indicated that this cDNA encoded hagfish MASP 1. This evidence strongly suggests that the hagfish defends itself against pathogens at least by the complement system composed of lectin pathway.     

Experimental studies on the light sense in the hagfish,Eptatretus burgeri andParamyxine atami (Cyclostomata)

Photo-reception and sensitivity to light were studied in two Japanese hagfishes,Eptatretus burgeri living in shallow water andParamyxine atami living in water of about 100m depth. Both species responded to general illumination by first moving the tail or head and then by swimming. Local illumination revealed that regions most sensitive to light were the skin of the tail in both species and a line of unpigmented skin running down the back ofE. burgeri. The light sensitivity of the lensless eyes, which are situated below the skin, was very weak in both species.P. atami showed shorter reaction time to light thanE. burgeri. No change in skin colour was induced either by almost complete hypophysectomy or by continuous illumination against a white background. Under-water observations with SCUBA revealed that free movingE. burgeri responded well to illumination uncovered during the night, but the ones buried in mud, with only the heads uncovered did not.     

Organization and evolution of highly repeated satellite DNA sequences in plant chromosomes

A major component of the plant nuclear genome is constituted by different classes of repetitive DNA sequences. The structural, functional and evolutionary aspects of the satellite repetitive DNA families, and their organization in the chromosomes is reviewed. The tandem satellite DNA sequences exhibit characteristic chromosomal locations, usually at subtelomeric and centromeric regions. The repetitive DNA family(ies) may be widely distributed in a taxonomic family or a genus, or may be specific for a species, genome or even a chromosome. They may acquire large-scale variations in their sequence and copy number over an evolutionary time-scale. These features have formed the basis of extensive utilization of repetitive sequences for taxonomic and phylogenetic studies. Hybrid polyploids have especially proven to be excellent models for studying the evolution of repetitive DNA sequences. Recent studies explicitly show that some repetitive DNA families localized at the telomeres and centromeres have acquired important structural and functional significance. The repetitive elements are under different evolutionary constraints as compared to the genes. Satellite DNA families are thought to arise de novo as a consequence of molecular mechanisms such as unequal crossing over, rolling circle amplification, replication slippage and mutation that constitute molecular drive.