The Actions of Enzymes on Lampbrush Chromosomes

1962 ◽  
Vol s3-103 (62) ◽  
pp. 173-203
Author(s):  
H. C. MACGREGOR ◽  
H. G. CALLAN
Keyword(s):  
Proteolytic Enzymes ◽  
Lampbrush Chromosomes ◽  
Triturus Cristatus ◽  
Trichloracetic Acid ◽  
Protease Digestion ◽  
Lateral Loop ◽  
Peptic Digestion ◽  
Rnase Digestion ◽  
Break Chromosome ◽  
Loop Matrix

The chromomeres of lampbrush chromosomes of Triturus cristatus are Feulgen-positive; they therefore contain DNA. After removal of their DNA in boiling trichloracetic acid, the chromomeres stain with fast green at alkaline pH; they therefore contain basic protein. The lateral loops are Feulgen-negative; they stain with toluidine blue at acid pH, but much less intensely following RNase digestion; they therefore contain RNA. The spheres of chromosomes V and VIII do not contain RNA. Unfixed lampbrush chromosomes retain a life-like appearance in 0.07 M K/NaCl at pH 6.2; in this medium the nuclear sap disperses. As pH is raised to 8.5 the matrices of lateral loops dissolve but chromosome axes remain unbroken. Above pH 8.5 lampbrush chromosomes dissolve. As pH is lowered from 6.2, at between 5.8 and 5.4 coagulation occurs. If pH is rapidly reduced still further, a persistent relaxed condition sets in between 2.5 and 2. In concentrations of K/NaCl above 0.5 M lampbrush chromosomes dissolve. Lateral loop matrices dissolve in 0.25 M K/NaCl but chromosome axes remain unbroken. In concentrations of K/NaCl below 0.05 M lateral loop matrices dissolve, but even in distilled water chromosome axes remain unbroken. Trypsin at pH 6.2 and at pH 7.8 strips the matrices from lateral loops and occasionally breaks matrix fusions. It causes chromomeres to swell and coalesce, but fails to break chromosome axes. The action of ‘pan-protease’ resembles that of trypsin in all respects. Pepsin at pH 6.2 strips the matrices from lateral loops, but does not destroy chromomeres. At low pH peptic digestion is slow: the enzyme is attacking coagulated chromosomes; but if peptic digestion precedes a lowering of pH the overall outcome is a rapid solution of loop matrix, and under these conditions matrix and sphere fusions are broken. If trypsin or ‘pan-protease’ digestion precedes a lowering of pH there is a similarly rapid solution of loop matrix; thus the action is not specifically referable to pepsin. Under no conditions does pepsin break the axes of lampbrush chromosomes. RNase at pH 6.2 strips the matrices from lateral loops; this action is detectable at extreme dilution. RNase does not destroy chromomeres, nor does it break chromosome axes. If tryptic digestion follows RNase digestion this too fails to break chromosome axes. Unlike the proteolytic enzymes and RNase, DNase at pH 6.2 breaks the fibril between adjacent chromomeres, and it also breaks the axes of lateral loops. Contrary to Mazia's experience with salivary gland chromosomes, versene does not break the axes of lampbrush chromosomes even when applied in media of low electrolyte concentration. These results indicate that uninterrupted fibres of DNA run throughout the lengths of lampbrush chromosomes.

scholarly journals Histone H4 acetylation and transcription in amphibian chromatin.

1993 ◽  
Vol 120 (2) ◽  
pp. 277-290 ◽  
Author(s):  
J Sommerville ◽  
J Baird ◽  
B M Turner
Keyword(s):  
Transcriptional Activation ◽  
Histone Deacetylation ◽  
Dense Matrix ◽  
Lampbrush Chromosomes ◽  
Loop Formation ◽  
Triturus Cristatus ◽  
Immature Oocytes ◽  
Intense Fluorescence ◽  
Histone H4 Acetylation ◽  
Lysine Residues

Lampbrush chromosomes from oocytes of the amphibian Triturus cristatus have been used to examine the role of histone acetylation in transcription by indirect immunofluorescence with antisera to H4 acetylated at specific lysine residues. Electrophoresis on acid-urea-Triton gels and Western blotting have confirmed the specificity of these antisera and defined the order in which particular lysine residues are acetylated in amphibian cells. As in mammals, lysine 16 is acetylated first, followed by 8 and/or 12 and then 5. With lampbrush chromosomes from immature (previtellogenic) oocytes, antisera to H4 acetylated at lysines 8, 12, and 16 labeled fluorescent foci at the bases of transcription loops. Antisera to H4 acetylated at lysine 5 labeled weakly (i.e., the tri- and tetraacetylated isoforms must be rare). Loops showed weak labeling of the chromatin axis but intense fluorescence at particular points, which probably represent incompletely decondensed chromatin. The RNP matrix of loops, including the RNP-rich sphere bodies and the dense matrix of "marker" loops, was not labeled. Treatment of immature oocytes with butyrate for 12 h to inhibit histone deacetylation did not affect immunolabeling, suggesting that turnover of H4 acetates is slow. In contrast, in chromosomes from mature oocytes, in which loops have retracted and transcription is low, butyrate caused an increase in labeling with all antisera, followed by the appearance of vestigial loops, weakly labeled, but with regions of intense fluorescence. These loops contain RNP and are presumably transcriptionally active. We conclude that H4 acetates turn over more rapidly in mature than immature oocytes and that histone hyperacetylation precedes, and possibly induces, loop formation and transcriptional activation.

In situ hybridization to lampbrush chromosomes: a potential source of error exposed

1980 ◽  
Vol 41 (1) ◽  
pp. 115-123
Author(s):  
H.G. Callan ◽  
R.W. Old
Keyword(s):  
Lampbrush Chromosome ◽  
Single Pair ◽  
Lampbrush Chromosomes ◽  
Triturus Cristatus ◽  
Rna Transcripts ◽  
Potential Source ◽  
Source Of Error ◽  
Simple Sequence ◽  
Do So

Denatured 3H-labelled DNAs containing Xenopus or human globin sequences hybridize to RNA transcripts on a single pair of lateral loops on lampbrush chromosome IX of Triturus cristatus carnifex, and to no other loops on this chromosome or the rest of the complement. However they do so, not because of the globin sequences in the probes, but rather because the plasmids from which the probes were prepared were constructed with G.C homopolymer tails. Simple sequence poly d(C/G)n probes also hybridize with RNA transcripts on this same pair of loops, and with no others.

scholarly journals ASPERMATOGENESIS, ANAPHYLAXIS, AND CUTANEOUS SENSITIZATION INDUCED IN THE GUINEA PIG BY HOMOLOGOUS TESTICULAR EXTRACT

1955 ◽  
Vol 101 (6) ◽  
pp. 591-604 ◽  
Author(s):  
Jules Freund ◽  
George E. Thompson ◽  
Murray M. Lipton
Keyword(s):  
Acetic Acid ◽  
Guinea Pig ◽  
Ammonium Sulfate ◽  
Guinea Pigs ◽  
Anaphylactic Shock ◽  
Proteolytic Enzymes ◽  
Trichloracetic Acid ◽  
Oil Emulsion ◽  
Highly Active ◽  
Water In Oil Emulsion

Guinea pig testicles were extracted with acetic acid; the extract was purified by removing material in consecutive precipitations with 30 per cent saturated ammonium-sulfate, trichloracetic acid, and chloroform. The solution so purified, when administered with complete adjuvants, was highly active in inducing impairment of spermatogenesis in guinea pigs. The activity resisted autoclaving at 15 pounds' pressure for 20 minutes, proteolytic enzymes, and formamide. Anaphylactic shock and cutaneous reaction to the purified homologous extract occurred in guinea pigs sensitized by the extract combined with adjuvants. For the production of aspermatogenesis it was essential to incorporate killed mycobacteria into the water-in-oil emulsion containing the antigen; but anaphylactic sensitization did not require the presence of mycobacteria.

Evidence For a Polarized Movement of the Lateral Loops of Newt Lampbrush Chromosomes During Oogenesis

1969 ◽  
Vol 5 (1) ◽  
pp. 1-25
Author(s):  
M. H. L. SNOW ◽  
H. G. CALLAN
Keyword(s):  
Rna Synthesis ◽  
Actinomycin D ◽  
Extreme Case ◽  
Full Recovery ◽  
Morphological Alteration ◽  
Lampbrush Chromosomes ◽  
Triturus Cristatus ◽  
Normal Morphology ◽  
Partial Inhibition

Actinomycin D inhibits RNA synthesis on the lateral loops of newt lampbrush chromsomes. Partial inhibition does not provoke marked morphological alteration of ordinary lateral loops, most of which recover to the full their capacity for RNA synthesis within 2 days of treatment. However, occasional ordinary loops do not recover completely within the first few days after treatment, and in such loops RNA-synthesizing capacity is restricted to a region adjoining the thinner insertion in the parent chromomere. A greater degree of inhibition of RNA synthesis is accompanied by loss of matrix from ordinary lateral loops, and in the extreme case the loop axes retract to their parent chromomeres and neighbouring chromomeres coalesce; for the ordinary loops, full recovery from this stripped condition is nevertheless possible. Some 20 µ per loop extends during the first day following exposure to actinomycin, and normal morphology and RNA-synthesizing capacity are regained within 2-4 days. The giant granular loop of Triturus cristatus cristatus chromosome XII responds to extreme actinomycin D poisoning in different fashion. Matrix does not at once slough off its loop axis, but the loop present at the time of treatment is progressively replaced by a new granular loop which develops between the parent chromomere and the original loop's dense tip. These observations support the theory that the DNA-containing axes of all lateral loops of lampbrush chromosomes continually extend from their parent chromomeres, engage in RNA synthesis while extended, and carry the associated RNP matrix along as they move towards the return insertions in the parent chromomeres, where loop axis retraction occurs.

Localization of histone gene transcripts in newt lampbrush chromosomes by in situ hybridization

1977 ◽  
Vol 27 (1) ◽  
pp. 57-79
Author(s):  
R.W. Old ◽  
G.H. Callan ◽  
K.W. Gross
Keyword(s):  
Dna Sequences ◽  
Histone Gene ◽  
Lampbrush Chromosomes ◽  
Rna Sequences ◽  
Psammechinus Miliaris ◽  
Lateral Loop ◽  
Gene Transcripts ◽  
Label Distribution ◽  
Chromosome I

Denatured 3H-labelled DNAs containing histone gene sequences originating from the echinoderms Psammechinus miliaris, Echimus esculentus and Strongylocentrotus purpuratus have been in situ hybridized to RNA transcripts on newt lampbrush chromosomes. Autoradiographs of the hybridized lampbrush preparations show labelling restricted to four or fewer lateral loop pairs all lying within the heteromorphic regions of chromosome I, also one or two loop pairs on chromosome VI, one loop pair on chromosome X and one loop pair on chromosome XI. For oocytes from a single newt, coincident label distribution is found with DNA's of diverse echinoderm origin; however different newts show some specific individual diversity in label distribution, including heterozygosity in the case of loops on bivalents VI and X. The more conspicuously labelled loops, particularly those on chromosome I, show a pattern of labelling which is explicable if the newt histone DNA sequences are confined to short intercepts of lateral loop axis. Transcription is initiated prior to the histone DNA sequences, proceeds through the histone DNA sequences, and beyond, and the histone RNA sequences are cut from the transcripts before the termination of transcription.

The role of Lampbrush Chromosomes in the formation of Nucleoli in Amphibian Oocytes

1965 ◽  
Vol s3-106 (75) ◽  
pp. 215-228
Author(s):  
H. C. MACGREGOR
Keyword(s):  
Phase Contrast ◽  
Nucleolar Organizer ◽  
Plethodon Cinereus ◽  
Oocyte Nucleus ◽  
Lampbrush Chromosomes ◽  
Triturus Cristatus ◽  
Amphibian Oocyte ◽  
Ambystoma Jeffersonianum ◽  
And Function

Theories concerning the mode of origin of peripheral nucleoli in amphibian oocytes have been examined and tested. In Triturus cristatus the giant fusing loops of the 3 shortest lampbrush bivalents resemble nucleoli when viewed in phase contrast and may be considered as possible sites of production of nucleoli. Giant fusing loops, however, differ from peripheral nucleoli in certain important respects, and animals lacking giant fusing loops on their lampbrush chromosomes nevertheless have normal peripheral nucleoli. Therefore, similarity in appearance between objects attached to lampbrush chromosomes and free peripheral nucleoli may not be significant. In oocytes of T. c. carnifex, T. c. karelinii, and T. c. danubialis, peripheral nucleoli do not increase in number during the lampbrush phase of oogenesis, except by division of pre-existing nucleoli towards the end of oogenesis. There are about 1,000 nucleoli per oocyte nucleus in each of these sub-species. In T. c. cristatus there are more nucleoli in large oocytes than in small ones, and it seems likely that in this sub-species the giant fusing loops add to the existing population of nucleoli in an oocyte by successively growing and shedding new nucleoli. A similar situation probably holds in Plethodon cinereus. Hexaploid oocytes from triploid females of Ambystoma jeffersonianum have 3 times as many nucleoli as diploid oocytes from diploid females of the same species. The number of nucleoli in an amphibian oocyte nucleus is therefore related to the number of sets of chromosomes in the cell. In yolky oocytes from hypophysectomized newts most peripheral nucleoli are firmly attached to the inner surface of the nuclear membrane; whereas in similar oocytes from unoperated or gonadotrophin-treated animals none of the nucleoli is so attached. On the basis of these observations 2 mechanisms are suggested for the formation of amphibian oocyte nucleoli. The first of these mechanisms probably operates in T. c. carnifex, where all peripheral nucleoli are formed before or soon after the chromosomes assume the lampbrush form, and no part of a lampbrush chromosome is involved in a process which adds to the existing population of nucleoli. The second mechanism probably operates in T. c. cristatus, where most of the peripheral nucleoli are formed before the lampbrush phase of oogenesis but a nucleolar organizer on the lampbrush chromosomes continues to grow and detach nucleoli throughout oogenesis. Both these mechanisms are discussed in terms of what is known of the chemical composition and function of peripheral nucleoli.

scholarly journals The influence of protease digestion and duration of fixation on the immunostaining of keratins. A comparison of formalin and ethanol fixation.

1986 ◽  
Vol 34 (8) ◽  
pp. 1095-1100 ◽  
Author(s):  
H Battifora ◽  
M Kopinski
Keyword(s):  
Monoclonal Antibody ◽  
Immunohistochemical Staining ◽  
Proteolytic Enzyme ◽  
Proteolytic Enzymes ◽  
Paraffin Sections ◽  
Optimal Length ◽  
Protease Digestion ◽  
Wide Range ◽  
Excellent Preservation ◽  
Formalin Fixed

The effect of three proteases--trypsin, pepsin, and pronase--on the immunohistochemical staining of keratins with a broad-spectrum monoclonal antibody was investigated in paraffin sections of formalin and ethanol-fixed tissues by means of the peroxidase-antiperoxidase method. Both the length of exposure to the fixative and the duration of proteolysis were varied over a wide range. Ethanol-fixed tissues showed excellent preservation of the antigenicity of keratins, and no appreciable differences in immunostaining related to the length of fixation were found. The use of proteolytic enzymes did not improve these results; on the contrary, it caused rapid tissue disintegration. Formalin-fixed epithelial tissues stained weakly or failed to stain unless they were treated with a proteolytic enzyme. The optimal length of proteolysis varied with the degree of fixation; tissues that were fixed for long periods of time in formalin required longer exposure to a proteolytic enzyme and were more resistant to digestion than were tissues that were fixed briefly. No significant advantage of one protease over another was found in this study. We conclude that a proteolytic step must precede immunostaining for keratins if the tissue is fixed in formalin, but that the digestion period must be adjusted according to the length of exposure to the fixative. The superiority of alcohol over formalin fixation for the preservation of the antigenicity of keratins is confirmed by this study.

Chicken Microchromosomes in the Lampbrush Phase: A Cytogenetic Description

2017 ◽  
Vol 152 (1) ◽  
pp. 46-54 ◽  
Author(s):  
Svetlana Galkina ◽  
Valerie Fillon ◽  
Alsu Saifitdinova ◽  
Aleksandra Daks ◽  
Maria Kulak ◽  
...  
Keyword(s):  
Lampbrush Chromosomes ◽  
Linkage Groups ◽  
Cytogenetic Mapping ◽  
Convenient Tool ◽  
Meiotic Chromosomes ◽  
Lateral Loop

Lampbrush chromosomes are giant, transcriptionally active, meiotic chromosomes found in oocytes of all vertebrates with the exception of mammals. Lampbrush chromosomes offer a convenient tool for cytogenetic mapping and, in particular, have been instrumental in mapping genes and linkage groups on chicken (GGA) chromosomes. Whereas cytogenetic maps of macrochromosome GGA1-10 and microchromosome GGA11-16 lampbrush bivalents have been established, identification and description of smaller microchromosome bivalents are still missing. In this work, we used specific FISH probes for the identification of 12 chicken lampbrush chromosomes formed by GGA17-28. Our observations on chromomere and lateral loop arrangement and chiasma position allowed us to construct the respective cytogenetic maps for these microchromosomes. For the 10 smallest chicken microchromosomes, GGA29-38, no individual molecular tags are available, yet they can be collectively marked using the PO41 repeat. The reported results contribute to building of working cytogenetic maps of the chicken karyotype.

Lampbrush chromosomes from semi-albino crested newts,Triturus Cristatus Carnifex (Laurenti)

1972 ◽  
Vol 28 (7) ◽  
pp. 856-860 ◽  
Author(s):  
G. Mancino ◽  
Irma Nardi ◽  
Matilde Ragghianti
Keyword(s):  
Lampbrush Chromosomes ◽  
Triturus Cristatus
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