Electrophoretic Banding Patterns Derived From Striated Muscle Samples Biology Essay

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In recent times fishes were identified mainly using the morphological characteristics. In the present time process of electrophoresis of serum proteins, sarcoplasmic proteins and liver protein are used to help in species identifying .Moreover number of enzymes are also used to identify the species. In fish 20 to 30% protein is accounted of total protein in the muscles.

To settle down the dispute of taxonomy in effective way in the fish and other organisms is unquestionable; especially when comparison between the species shows isoenzyme patterns shows diagnostic loci. A certain variable percentage of loci has detected the correct identification of closely related units.

To identify and delimit the fish stock, isoenzyme are used as molecular genetic-markers in effective ways. They are also used in other living stock. Heterogeneity among the population samples can be estimated by using the statistical data. To differentiate the populations of several fish species, process of polymorphism is successfully used. Failure of the present study to diagnose the different species of the fish on the basis of their loci, which can be used to distinguish these two species and polymorphic loci does not rule out their existence in other enzyme systems.

Both molecular markers and chromosomes are frequently used to determine the taxonomic status of the fish.

A process of denaturation is found there. Protein’s secondary, tertiary, and quaternary structure prior to electrophoresis using heat and SDS is disrupted. This results in having only the linear chain of amino acids (the primary structure) to migrate through the matrix. In this lab, the Laemmli sample buffer, which contains SDS, will be used to denature the proteins

Methods and RESULTS

Samples of the fish thread herring were taken from the different locations of Ocean Arabia. All samples were frozen and shifted to the relevant laboratory. They were stored at the ultra cold freeze at -60C0 to -70C0. These samples were kept there for few days. After that they were taken from there and handed over to team for the analysis. Their first gill arches were removed to count the gill-raker. To keep the identity for every species a numbered label was attached to a gill cover. Again the fish were stored in the ultra cold until the next step of analysis to be taken. Around 100 of these fish samples were classified by using the gill raker-standard length tabular key. For 10 fish gill-raker were not obtained. Of these 9 were assigned on the basis of allozyme phenotypes and it was found that these were congruent with the gill-raker diagnoses of 100 fish. One fish was inferred to be a hybrid between 0. bulleri and 0. Medirastre.Six collections of the species have the following composition. Bahia Magdalena (7 0.medirastre, 2 0. libertate); Guaymas (all 0. libertate);Calafia 1 (4 0. bulleri, 6 0. medirastre, 37 0.libertate, and 1 hybrid); Calafia 2 (6 0. bulleri, 50. medirastre, 33 0. libertate); Calafia 3 (2 0. bulleri,7 0. medirastre, 36 0. libertate); and Hapemsa ZZZ (all 0. bulleri)Summing the collections, 56 0. bulleri, 25 0. medirastre, 129 0. libertate, and 1 hybrid were obtained.

Process of Electrophoresis involves to keep the samples in labeled plastic trays with the equal volume of the buffer 0.5M Tris-HC1 pH 7.1. Genetic interpretation of Zymograms and Methods for horizontal starch-gel electrophoresis, protein assays were conducted and result were the same as we expected. The procedure used to separate and resolve 19 enzymes and protein is given in the following table.

Table: Illustrates the Starch-Gel Electrophoretic Protocol

Sr #

Enzyme or protein

E.C No

Tissue

Buffer

No of loci

01

Aspartate aminotransferase

2.6.1.1

E.L

B

1

02

Triosephosphate isomerase

5.3.1.1

M

B.F

1

03

Alcohol dehydrogenase

1.1.1.1

E

F

1

04

Superoxide dismutase

1.15.1.1

L

B

1

05

Esterase

Non-specific

L

E

1

06

General proteins

Non-specific

E+M

A

8

07

Fumarate hydratase

4.2.1.2

M

C,D

1

08

6-phosphogluconate dehydrogenase

1.1.1.44

E,L

C,D

1

09

Glucose-6-phosphate isomerase

5.3.19

M

B

1

10

Phosphoglucomutase

2.7.5.1

M

A

1

11

Glyceraldehyde-3-phosphate dehydrogenase

1.2.1.12

E,L,M

C,D

1

12

L-phenylalanyl-proline

3.4.13.9

E

B

1

13

Isocitrate dehydrogenase

1.1.1.42

L

D

1

14

L-leu-val + L-leu tyr

3.4.13.11

E

B

2

15

Lactate dehydrogenase

1.1.1.27

E,L,M

B

3

16

L-leu-gly-gly

3.4.13.11

E

B

1

17

Malate dehydrogenase

1.1.1.37

M

F

1

18

Malic enzyme

Di- and tri-peptidases

1.1.1.40

H

F

1

19

L-gly-leu

3.4.13.11

E

B

1

Fig2: Illustration of the process of SDS-PAGE Analysis

On the basis of these tests and experiments a table containing the facts of the ratio and percentage are shown in the table. This table provides the optimistic statistics of our work.

Number of Bands

(A)9

(B)8

(C)7

(D)6

(E)5

(F)4

(G)3

(H)2

(I)1

Number found in Pop.

18

16

15

1

10

9

4

22

5

Frequency in Pop.

0.18

0.16

0.15

0.01

0.1

0.09

0.04

0.22

0.05

Frequency in Pop (%)

185

16%

15%

1%

10%

9%

4%

22%

5%

Table: Showing the number of bands, number found in population, frequency in population and frequency % in population.

Statistics

For statistics analysis individual were grouped into the nine population samples.

(1) 5 0. bulleri pooled from the Culufiu samples.

(2) 22 0. bulleri from the Hupemsu IZZ sample.

(3) 4 0. medirustre from Bahia Magdalena.

(4) 9 0. medirustre pooled from the Culujia samples.

(5)10 0. libertute from Guaymas.

(6) 10. libertute from Bahia Magdalena.

(7), (8), and (9) 15, 16 and 18 0. libertute from Culujiu samples 1,2, and 3, respectively.

No evidence was found for genetic heterogeneity among the small collections obtained form the population 1 and 4.

By testing the heterogeneity of allelic frequencies, samples were pooled for the measurement of interspecific genetic similarities and distances. Estimation of genetic distance was calculated by using the averaging (UPGMA) in the CLUSTER. By pooling of the rare alleles absolute frequencies were calculated in the population samples of 0 Libertute.

Analysis of results

Gill Racker counts: Gill racker counts for the 99 of the 110 clearly falls into the three groups as these groups are described previously.

Lactate dehydrogenase: Aim of our study is to know the phenotype frequencies of enzyme lactate dehydrogenase of the population of the species of Herry.Two major Electrophoretic bands of Lactate dehydrogenase were observed with an isoenzym pattern. They were called as Ldh1 and Ldh2. These are shared by the all discussed species. Question comes in mind that how far they are helpful as taxonomic genetic markers for the two species. The Ldh1 was found near the anodic region of the gel while Ldh2 was seen near the cathode region. However the presence of these bands does not prevent identifying the correct location of these enzymes. However these three species in our study are clearly distinct but amount and variation in allozyme frequencies are still to be in consideration. This Allozyme data give the verification of the geographical variation.

Differences among the species are found on the basis of the allozyme.

Marphometric measurement shows the correlation in the all 19 traits is found. This Marphometric variation shows the variation in body size of the species.

Conclusion

Three species of the thread herrings have been given special consideration in our study work. These three species are described on the basis of difference found in number of gill-rakers. Taken the geographical variation and the standard length into account, it is asked about the validity of these species. Due to this reevaluation of the taxonomic status is sought. Electrophoretic and multivariate Marphometric study verified that accurate gill-raker numbers on the basis of the bio-chemical genetic evidence. Our study has suggested that external morphological characteristics are involved to discriminate the species of fish. Our work has revealed about the evolution of three species.

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