Intrinsic factors influence the attachment of fragments of the green alga Caulerpa filiformis

Asexual reproduction via fragmentation is an integral component of the life history of many marine organisms. In south-eastern Australia the green macroalga Caulerpa filiformis (Suhr) Hering is abundant on many rocky shorelines and fragments are very abundant in the water column. We examined some of the factors that may influence the growth and successful reattachment of fragments of C. filiformis that potentially contribute to its success. Field surveys revealed that C. filiformis fragments were of variable morphologies (from small fronds to entire thalli) and sizes (0.1-60 cm in length). All fragments of C. filiformis were viable propagules that survived and grew well in the laboratory. However, some fragments grew more than others, in particular larger sizes (7.5 cm class) of certain morphologies (fragments that consisted only of a single frond). Rhizoids were produced by all fragments, but again, larger fragments produced more rhizoids than smaller fragments. The force required to detach fragments was proportional to the number (but not size) of rhizoids, demonstrating the importance of rhizoids for attachment. The rate of attachment of fragments was also fast, usually within 12 h, but the longer these fragments were attached to a substratum the greater the force required to dislodge them. Interestingly, fragments only produced rhizoids if they were resting on a substratum which may explain the small proportion of fragments with rhizoids in the field (< 5%). Ultimately, the long term success of C. filiformis fragments was low as only 1.6% of fragments attached within 24 h and none of these persisted for longer than 2 days. Nonetheless, the abundance and viability of fragments of C. filiformis available to attach suggest that asexual fragmentation is a successful reproductive strategy in this seaweed.     

Physical map of the bacteriophage T5 genome based on the cleavage products of the restriction endonucleases SalI, SmaI, BamI, and HpaI.

A physical map of the bacteriophage T5 genome was constructed by ordering the fragments produced by cleavage of T5 DNA with the restriction endonucleases SalI (4 fragments), SmaI (4 fragments), BamI (5 fragments), and HpaI (28 fragments). The following techniques were used to order the fragments. (i) Digestion of DNA from T5 heat-stable deletion mutants was used to identify fragments located in the deletable region. (ii) Fragments near the ends of the T5 DNA molecule were located by treating T5 DNA with lambda exonuclease before restriction endonuclease cleavage. (iii) Fragments spanning other restriction endonuclease cleavage sites were identified by combined digestion of T5 DNA with two restriction endonucleases. (iv) The general location of some fragments was determined by isolating individual restriction fragments from agarose gels and redigesting the isolated fragments with a second restriction enzyme. (v) Treatment of restriction digests with lambda exonuclease before digestion with a second restriction enzyme was used to identify fragments near, but not spanning, restriction cleavage sites. (vi) Exonucleases III treatment of T5 DNA before restriction endonuclease cleavage was used to locate fragments spanning or near the natural T5 single-chain interruptions. (vii) Analysis of the products of incomplete restriction endonuclease cleavage was used to identify adjacent fragments.     

A physical map of the genome of temperate phage phi 3T.

A physical map of the genome of Bacillus subtilis bacteriophage phi 3T was constructed by ordering the fragments produced by cleavage of phi 3T DNA with restriction endonucleases AvaII (2 fragments), BglI (2 fragments), SmaI (3 fragments), BamHI (6 fragments), SalI (7 fragments), AvaI (7 fragments), SacI (12 fragments), PstI (14 fragments), and BglII (26 fragments). Two techniques were used to order the fragments: (1) Sets of previously ordered restriction fragments were isolated and redigested with the endonuclease whose cleavage sites were to be mapped. (2) Fragments located near the ends of the genome or near the ends of other restriction fragments were ordered by treating the DNA with lambda exonuclease prior to restriction endonuclease cleavage. The susceptibility of phi 3T DNA to 15 other restriction endonucleases is also reported.     

Ultraviolet-sensitive ooplasmic factors are responsible for the development of an endoderm-specific alkaline phosphatase in the ascidian embryo

The ascidian egg contains muscle and endoderm determinants that play critical roles in the specification of muscle and endoderm cells, respectively. Endoderm cells of the ascidian embryo express alkaline phosphatase (AP) as a tissue-specific enzyme. We obtained egg fragments from the unfertilized eggs of Ciona savignyi by means of centrifugal force. The largest fragment (red fragments) contained the egg nucleus while other small fragments (black, clear and brown fragments) were anucleate. When inseminated, only red fragments developed into partial embryos, which showed only epidermis cell differentiation and, very rarely, AP activity. When red fragments were fused with other fragments, only black fragments promoted AP expression, suggesting that endoderm determinants were concentrated in the black fragments. A lower dose (1500 J/m²) of ultraviolet (UV) light did not eliminate the AP-promoting ability of black fragments, while this dose significantly repressed the ability to promote the expression of the muscle-marker. A higher dose (4500 J/m²) of UV light markedly reduced the AP-promoting activity of black fragments. These results suggest that factors for endodermal AP development are inactivated by UV irradiation, but are more resistant than muscle determinants.     

Differences in the catalytic properties of fragments of the ceruloplasmin molecule

Human ceruloplasmin (Cp) molecule is split into fragments by a contaminating protease upon storage of enzyme preparations. These fragments were separated by SDSPAAG electrophoresis and their Mr were estimated. Separate fragments were subjected to immunoelectrophoresis in agarose gel containing rabbit antibodies to human Cp. The immunoprecipitation peaks were then specifically stained to reveal the oxidase activity of the fragments towards o-dianisidine and L-cysteine. All the fragments were able to oxidize the latter, however, only the whole Cp molecule and the two of its largest fragments could oxidize the former. It seems likely that oxidation of L-cysteine does not require the presence of several copper ions constituting the catalytic centre of the blue oxidase (Cp.). contrarily, o-dianisidine seems to be oxidized by the multicopper active site of the enzyme rather than by the autonomously acting singular copper(s). Since o-dianisidine is oxidized by the fragments of Cp lacking the C-terminal polypeptide, which was thought to bind all the coppers of the active centre, it was assumed that some of the latter are bound by amino acids located in another part of the molecule.     

A study of DNA depolyploidization and depolytenization of the heterochromatized gonosomal chromatin bodies in the secondary giant trophoblast cells of the field vole Microtus rossiaemeridionalis using cytophotometry

A study was made of the distribution of the heterochromatized gonosomal chromatin bodies (GCB) material in the course of nuclear fragmentation of secondary giant trophoblast cells resulting in polykaryocyte formation at the late stage of their differentiation. A simultaneous DNA cytophotometry in GCBs and nuclear fragments showed a progressive GCB DNA content decrease proportional to that of DNA content in nuclear fragments. DNA contents in the nuclear fragments corresponded to 2c, 4c and 8c. In most cases 1-2 GCBs were found in the nuclear fragments of different ploidy levels. Both the total DNA content in GCBs and the DNA content in separate GCBs well correlated with the ploidy levels of fragments. The data obtained demonstrate a regular, whole-genome distribution of chromosomal materials into the nuclear fragments exemplified by sex chromosome distribution in compliance with the ploidy of nuclear fragments. We discuss a possible mechanism of nuclear fragmentation that may ensure substantially a balanced genome of nuclear fragments without leading to mitotic cycle renewal in the giant trophoblast cell population.     

Characterization of the isolated 20 kDa and 50 kDa fragments of the myosin head

We have isolated two proteolytic fragments of subfragment 1 (S-1) of myosin from rabbit skeletal muscle. These fragments, identified by their molecular weights of 20 and 50 kDa, may be functional domains that, when isolated, retain their specific function. We have studied several structural and functional features of the 20 and 50 kDa fragments. Considerable secondary structure in both fragments has been observed in CD spectrum studies. Previously CD spectra showed 64% ordered structure for the 20 kDa fragment (Muhlrad and Morales, M.F. (1984) Proc. Natl. Acad. Sci. 81, 1003) and here we show 71% ordered structures for the 50 kDa fragment. Fluorescence lifetime studies of tryptophan residues in the 50 kDa fragment and 1,5-IAEDANS-labeled SH-1 in the 20 kDa fragment are used to investigate the tertiary structure of the fragments. We find the tertiary structure relating to this measurement of both fragments to be intact; however, the reaction of 1,5-IAEDANS with SH-1 on the isolated 20 kDa fragment is less specific than with S-1. Furthermore, the fragments showed a tendency to aggregate. The domain concept of S-1 was supported by the characteristic biochemical function of the isolated fragments. Both of the fragments were effective in competing with S-1 for binding to actin in acto-S-1 ATPase measurements. From these studies and in direct binding measurement the 20 kDa fragment proved to bind with higher affinity to actin than did the 50 kDa fragment.     

Alignment of biologically active domains in the fibronectin molecule

Gelatin-binding material was isolated from a human plasma cryoprecipitate by affinity chromatography on gelatin-Sepharose. Individual fragments of fibronectin with Mr = 170,000, 100,000, and 80,000 and a mixture of fragments with Mr = 205,000 and 190,000 (200K fraction) were isolated from this material. These fragments reacted with antifibronectin and with antibodies to a gelatin-binding Mr = 70,000 tryptic fragment of fibronectin. They all shared the same NH2-terminal amino acid sequence. The 205K and 190K fragments bound also to heparin-Sepharose, whereas the smaller fragments did not. The 200K fraction and the 170K fragment mediated cell attachment when used to coat plastic, whereas the 100K and 80K fragments were inactive in this assay. Further digestion of the 205K and 190K fragments with chymotrypsin yielded separate sets of smaller fragments that bound to either gelatin-Sepharose or heparin-Sepharose, as well as fragments that did not show either of these binding activities but mediated cell attachment. Since the NH2-terminal ends of the 205K, 190K, 100K, and 80K fragments are the same, the results define the order of the active sites in the fibronectin molecule as gelatin-binding site, cell attachment site, and heparin-binding site.     

Cleavage map of the simian-virus-40 genome by the restriction endonuclease III of Haemopholus aegyptius.

Enzymic digestion of Simian virus 40 (SV40) DNA with Haemophilus aegyptius restriction endonuclease Hae III results in 10 major and eight minor fragments. These were resolved by electrophoresis on graduated polyacrylamide slab gels. All fragments have been characterized with respect to the size relative to the Haemophilus influenzae Rd fragments (Hind). They were ordered on the SV40 DNA map by means of overlap analysis of the double cleavage products derived from sequential digestion of Hind fragments with Hae III endonuclease and Hae fragments with Hind II + III enzyme, as well as by other reciprocal cleavage experiments, including those involving Haemophilus para-influenzae fragments. In this way the 18 Hae III cleavage sites and the 13 Hind sites have been localized on the circular SV40 DNA map.     

Gray and Red Fragments of the Egg of the Ascidian Ciona savignyi: Preferential Development of Muscle Cells from Gray Fragments

The egg of the ascidian Ciona savignyi is pinkish red with brownish myoplasm that contains the putative determinants responsible for differentiation of muscle cells. When dechorionated unfertilized eggs were centrifuged at moderate speed, eggs were divided into centripetal, small gray fragments and centrifugal, large red fragments. The former contained the female pronucleus and clear cytoplasm, while most of the latter was filled with yolk granules. An antibody raised against the myoplasm of C. intestinalis eggs extensively stained the cortical region of gray fragments, while the antibody stained only small regions of the red fragments. After insemination, both fragments cleaved and gave rise to partial embryos. When development of muscle and epidermal cells in the partial embryos was examined with specific antibodies, muscle development was conspicuous in gray partial embryos, while epidermal differentiation was extensive in red partial embryos. Furthermore, when expression of markers of differentiation was examined in cleavage-arrested gray and red fragments, the number of arrested gray fragments exhibiting the muscle marker was about three-fold greater than in controls. These results suggest that putative muscle determinants are concentrated into gray fragments.     

Restriction enzyme analysis of the Plasmid ColIb DNA

Summary Plasmid ColIb (61.5 Mdal) was digested with restriction enzymes EcoRI and HindIII. The DNA digestion products were separated by electrophoresis on 1.2% agarose gels. There were identified 22 fragments of ColIb DNA generated by the endonuclease EcoRI and 21 fragments produced by HindIII. Molecular weights of the fragments were estimated. The total molecular weight of the fragments generated by EcoRI was 61.42 Mdal and for HindIII fragments 62.79 Mdal.     

Cloning of DNA fragments of the genetic transfer factor pAP39

Large HindIII digested fragments of the plasmid pAP39 have been cloned on the cosmid vector pHC79. The study of the structure of HindIII fragments of plasmid pAP39 in the recombinant plasmids has shown that these fragments are represented by f1 + f2 fragments from the plasmid pAP1055, by f1 + f6 fragments from the plasmid pAP1056, by f2 + f3 fragments from the plasmid pAP1057 and by two f3 fragment from the plasmid pAP1058. Physical maps of the recombinant plasmids have been constructed. The plasmid pAP39 is shown to contain two functionally active tra regions.     

Position-specific interaction between cells of the imaginal wing and haltere discs of Drosophila melanogaster.

When fragments of the imaginal wing disc from opposite ends of the disc are mixed prior to culture, intercalary regeneration occurs so that structures are produced which neither of the fragments would have produced if they had been cultured alone. I report here that fragments of the imaginal wing and haltere disc interact in a position-specific way. Mixing of homologous fragments does not result in regeneration, while mixing of fragments from opposite ends of the discs does. Thus the interaction of wing and haltere disc fragments shows the same positional specificity as the mixing of two wing fragments.     

Conformational and ligand binding properties of the isolated domains from the beta 2 subunit of Escherichia coli tryptophan synthetase investigated by the reactivity of their cysteines.

A mild proteolytic treatment of the dimeric beta 2 subunit of Escherichia coli tryptophan synthetase (L-serine hydrolase (adding indole) EC 4.2.1.20) is known to nick each polypeptide chain into two complementary fragments, F1 and F2 (H?gberg-Railbaud, A., and Goldberg, M.E. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 442-446). The reactivity of the cysteines in the isolated or associated fragments is studied and used to characterize the structural and functional properties of these fragments. It is shown that the total number of cysteines, their reactivity to dithiobisnitrobenzoate, and their protection by various ligands are the same in the nicked and intact enzyme, thus demonstrating the close structural analogy between these two proteins. In the isolated F1 fragments two cysteines are reactive and two are buried, thus confirming that this fragments has a compact, globular structure. Various ligands tested fail to produce any modification of the cysteines in the isolated fragments, thus suggesting that none of the fragments alone carries a binding site for the substrates and coenzyme.     

Morphological characterization of the asexual reproduction in the acorn worm Balanoglossus simodensis

The acorn worm Balanoglossus simodensis reproduces asexually by fragmentation and subsequent regeneration from the body fragments. We examined the morphogenesis of its asexual reproduction. At first, we collected asexually reproducing specimens and observed their morphogenesis. Then, we succeeded in inducing the asexual reproduction artificially by cutting the worm at the end of the genital region. The process of morphogenesis is completely the same between naturally collected and artificially induced specimens. The stages during morphogenesis were established on the basis of the external features of the asexually reproducing fragments. The internal features of the fragments were also examined at each stage. In a separate phase of the study, the capacity for regeneration of some body parts was also examined by dividing intact worms into about 10 fragments. Although the capacity for regeneration varied among the different body parts, some fragments regenerated into complete individuals in 1 month. The process of regeneration was the same as that in the asexually produced fragments.     

Gene localization, size, and physical map of the chromosome of Streptococcus pneumoniae.

A physical map of the Streptococcus (Diplococcus) pneumoniae chromosome, which is circular and 2,270 kbp in circumference, has been constructed. The restriction enzymes ApaI, SmaI, and SacII were used to digest intact chromosomes, and the fragments were resolved by field inversion gel electrophoresis (FIGE). The digests produced 22, 20, and 29 fragments, respectively. The order of the fragments was deduced from Southern blot hybridization of isolated labeled fragments to separated fragments of the various restriction digests. Genetic markers were correlated with the physical map by transformation of recipient cells with FIGE-isolated DNA fragments derived from genetically marked S. pneumoniae strains. In addition, markers were mapped by the hybridization of cloned genes to FIGE-separated restriction fragments. Six rRNA gene (rrn) clusters were mapped by hybridization to rrn-containing fragments of Haemophilus influenzae.     

Genetics of Euglossini bees (Hymenoptera) in fragments of the Atlantic Forest in the region of Vi?osa, MG.

With uncontrolled deforestation, forest fragments remain, which in most cases are in different stages of regeneration and present isolated populations. In the present study we analyzed the genetic patterns of Eulaema nigrita populations in seven Atlantic Forest fragments of different sizes and successional stages in the region of Vi?osa, MG. This was done by RAPD molecular markers. We observed that the area of the fragments had no effect on the genetic variability of E. nigrita in the direction predicted by meta-population models. Medium-sized well-preserved woods presented the lowest variability, whereas large and small woods were statistically identical. The evidence supports the notion that rural areas present greater dispersal among fragments, implying greater similarity between the populations of fragments located in rural areas when compared to fragments in urban areas.     

Preliminary study on the detection of the SARS-CoV specific target cDNA fragments by multiplex PCR

The multiplex polymerase chain reaction (PCR) technique was applied to detect the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) specific target cDNA fragments in the present study. The target cDNA fragments of SARS-CoV were synthesized artificially according to the genome sequence of SARS-CoV in GenBank submitted by The Chinese University of Hong Kong, and were used as simulated positive samples. Five primers recommended by World Health Organization (WHO) were used to amplify the fragments by single PCR and multiplex PCR. Three target cDNA fragments (121, 182 and 302 bp), as well as the three different combinations of any two of these fragments, were amplified by single PCR. The combination of these three fragments was amplified by multiplex PCR. The re~sults indicated that the multiplex PCR technique could be applied to detect the SARS-CoV specific target cDNA fragments successfully.     

XbaI, PstI, and BglII restriction enzyme maps of the two orientations of the varicella-zoster virus genome.

Cleavage of varicella-zoster virus DNA with the restriction endonucleases PstI, XbaI, and BglII resulted in 18, 22, and 20 fragments, respectively. Based on the molecular weights and molarities of these fragments, a molecular weight of 84 x 10(6) could be calculated for the varicella-zoster virus genome. In both the XbaI and the BglII patterns, four 0.5 M fragments were identified. The arrangement of the fragments was determined by molecular hybridization techniques, and the terminal fragments were identified by lambda exonuclease digestion. The 0.5 M fragments, of which two were located at the same terminus of the genome, contained repeated sequences: one terminally and one inverted internally. These results were in agreement with the existence of two equimolar subpopulations of the varicella-zoster virus genome, differing in the relative orientation of a short region of unique sequences. This region was bounded by the repeated sequences. From the molecular weights of the submolar fragments, a maximal molecular weight of 5 x 10(6) for the repeated region and a minimal molecular weight of 3.5 x 10(6) for the short unique sequence could be calculated.     

Coral reef restoration in the Eastern Tropical Pacific: feasibility of the coral nursery approach

Due to the worldwide degradation of coral reefs, the active restoration of these ecosystems has received considerable attention in recent decades. This study investigated (1) the feasibility of using coral nurseries for restoration projects, (2) the minimum size required for a Pocillopora damicornis (Pocilloporidae) coral fragment to survive and grow in a nursery, and (3) the optimal transplant size of a fragment when transplanted to a degraded reef at Gorgona Island (Colombian Pacific). For this investigation, 230 fragments were transplanted directly to El Remanso reef, and another 150 fragments were maintained in in situ nurseries. Every 2 months, the length, weight, and survival of the fragments were recorded. After growing for 134 days in the nurseries, the 52 surviving fragments were transplanted to El Remanso reef, and after 5 months, the same variables were measured. Among the nursery‐reared fragments, the largest (4 to <8 cm) had the highest survival and growth rates, whereas among the directly transplanted fragments, the smallest fragments (<2 cm) had the highest survival and growth rates. However, the nursery‐reared fragments acquired greater structural complexity (arborescent morphology), and they were all alive 156 days after transplantation and presented a maximum linear growth rate of over 2 cm, which was higher than that of the directly transplanted fragments. Apparently, the arborescent morphology acquired during the nursery period provides advantages to the colonies that favor greater success when transplanted. Therefore, nursery‐reared fragments of P. damicornis between 2 and 4 cm are the most appropriate for use in restoration projects.