Lampbrush Chromosomes

They belong to a class of specialized chromosomes discovered in the oocytes of animals except for mammals. Walter Flemming discovered these specialized structures. They show the presence of highly extended regions. The tailed and the tailless amphibians, birds, and insects show these specialized chromosomes. These chromosomes do not occur in plants. The geneticists visualize the lampbrush chromosomes even under the light microscope. Hence, they involve an easy staining procedure. The lampbrush chromosomes exist as bivalents, each having two sister chromatids. These chromosomes get visualized mainly in the diplotene stage of prophase I of meiosis-l. During this stage, the chromosomes get converted into lampbrush shaped. Meaning, they resemble a brush for cleaning the chimneys. They consist of loops occurring in pairs. They occur as one on either side of the axis. These chromosomes involve active transcription. Mainly, the loops involve vigorous RNA synthesis. The basic units of lampbrush chromosomes include a pair of deoxyribonucleoprotein (DNP) fibers. They run along the axis of the chromosomes. However, the centromere of the lampbrush chromosome lacks the lateral loops. It is known as a chromomere. It gets flanked by the condensed regions showing the axial bars. 
The axial bars include the pericentromeric heterochromatin. These regions show the presence of highly repetitive satellite DNA. Thus, it does not involve any looped regions. The lampbrush chromosomes do not occur in the males. However, exceptions include Drosophila Y chromosomes showing lampbrush loops. These regions involve active transcription process. The lampbrush chromosomes show a greater thickness as compared to the 30nm chromatin fiber. It is due to the high amount of ribonucleoprotein arising due to active transcription. The amphibian lampbrush chromosomes show the presence of telomeres. These regions look like granules. However, the telomeres of the bird lampbrush chromosomes consist of additional loops attached to the telomeric granules at one end. The other end carries a free telomeric sequence involving transcription from only the C- rich strand.

The homologous loops differ in their size and structure. The lampbrush chromosomes involve uninemy of the chromosomes. Meaning,  they consist of one DNA duplex.

Image: Lampbrush Chromosomes

Transcription process in the loop:

Attachment of the RNA polymerase enzyme to the principal axis of the loop gets revealed through the electron microscopy. The RNA fibrils start extending upon the action of RNA polymerase enzyme. These fibrils go on increasing their length. The hnRNA or the heterogeneous nuclear RNA covers different kinds of RNA molecules possessing different lengths. These molecules are known as the precursors for mRNAs in the eukaryotic cells. The loops mainly perform the transcription of the hnRNAs. The structure of the lateral loop consists of the asymmetrical matrix consisting of RNA transcripts. The hnRNA transcripts also consist of their respective binding proteins. The synthesis of RNA starts at the thinner end. It gets completed at the thicker end. Ultimately, the ribonucleoprotein gets released. Important components of the cell involved in the structure of the lampbrush chromosomes are known as actin filaments. Actin is a kind of protein possessing filaments. 
The loop gets extended from the chromomeric axis with the help of actin filaments. In the end, the loops disappear. The transcriptional regions in the lampbrush chromosomes include lateral loops. An important structure of the lampbrush chromosome known as chromomere plays a crucial role here. The chromomere is the axial condensate of the lampbrush chromosome. It appears like that of a bead. The paired loops extend from the chromomere. The composition of the chromomere includes tight packaging of the DNA with histones forming arrays of deoxyribonucleoprotein. The newt chromosomes consist of 104 lateral loops in one haploid complement. Thus, lateral loops are easy to identify. They possess distinct units with different morphologies. They also act as the units of inheritance. Since they get involved in the transcription process, they are known as the units of transcription.

Staining lampbrush chromosomes:

Silver staining technique involves staining the objects of study using silver. It is possible to stain the lampbrush chromosomes with silver staining. Alternatively, they get stained with antibodies against the RNA processing proteins. Actively expressing regions become clearly visible under the fluorescence microscope. The RNA splicing machinery appears like round-shaped granules under the microscope.

The procedure for preparing lampbrush chromosomes:

The procedure worked well firstly in the urodele amphibian oocytes. The first step involves culturing the actively dividing tissues or cells extracted from the organism. Next, the cultures get processed to obtain only the oocytes. The nucleus of the oocytes gets extracted removing the membrane. Next, the lampbrush chromosomes get freed up from the nucleus of the oocyte. These chromosomes get fixed on the coverslip for microscopic examination.

Consequences of stretching the lampbrush chromosomes:

The lampbrush chromosomes consist of highly extended regions. Upon stretching these chromosomes, the breaks occur in them. These breaks occur transversely. The chromosomes thus held together with the help of fibers, thereby forming loops. The looped regions consist of one double-stranded DNA molecule. The axis regions consist of two double-stranded DNA molecules. Digestion of the lampbrush chromosomes with DNase-I creates two adjacent cuts. The rate of production of breaks shows a direct relationship with the square of the length of the time of digestion. Two double-stranded DNA molecules present in the axis have a different equation. The rates of production of breaks in them show a direct relationship with the digestion time raised to the power of four.

Drosophila lampbrush chromosomes:

The lampbrush chromosomes in Drosophila also show the presence of loops. However, they undergo different mechanisms. The loops consist of simple sequences. They show highly repetitive DNA, retrotransposons, and middle repetitive DNA sequences. All of them get transcribed. Out of many, an at least one loop thread out and shows the genes encoding Dynein. It forms the outer arms of the microtubules in the sperm tails. The loops show relatively larger sizes. The reason includes the presence of high amounts if intronic sequences. Male Drosophila lampbrush chromosomes do not exhibit crossing over during meiosis. Thus, they do not have any mechanisms to prevent rapid growth of clusters of the satellite DNA.

The significance of lampbrush chromosomes:

These chromosomes mainly play a role in the transcription process. Apart from this, lampbrush chromosomes get involved in the maternal gene expression. During oogenesis, the lampbrush chromosomes provide important clues regarding the sequence expression. The maternal mRNA synthesis mainly occurs in the oocytes. It helps the early embryos to get the mRNA from the maternal part. Thus, they serve in the embryonic development.

References:

[1] Lampbrush Chromosomes, Harold G. Callan

[2] Chromosomes: Organization and Function, Chapter 14, Adrian T. Sumner

[3] Principles of genetics, 8th edition, Page 134, Gardner, M. J. Simmons, D. P. Snustad

Copyright, 2019 All Rights Reserved

What is cytogenetics?

It is a branch of genetic involving the study of the cell and the chromosomes in detail. The chromosomal studies include the grouping and numbering of chromosomes, studying their structures, their variations in numbers, and the related conditions. It involves the behavior of the chromosomes during the mitotic and the meiotic phases. Studying chromosomes involves various techniques. Conventional cytogenetic techniques include karyotyping and banding techniques. Molecular cytogenetic techniques involve modern tools to detect chromosomal aberrations. FISH, Array-CGH, and many others help in studying the chromosomes.

The human cytogenetics relates to the study of human chromosomes and the mechanisms and anomalies associated with the same. The plant and animal cytogenetics cover the study of the respective chromosomes and the conditions associated with the structural and numerical variations. The human chromosomes involve autosomes and sex chromosomes. The total complement of chromosomes includes 46 number of chromosomes. The study of every chromosome in detail includes revealing its structure, the banding pattern, the genes in every chromosome, the conditions associated with same, and overall functioning of the chromosome.

The human cytogenetics involves the study of human chromosomes and the anomalies associated with the same. Let us discuss the human chromosomes in detail.

Image 1: Chromosomes and cytogenetics

Human chromosomes:

The chromosomes occur in the nucleus. They consist of DNA compacted with histone proteins. The word chromatin indicates the strands of the chromosomal material in the interphase. It shows the presence of coiled and extended regions. There are two types of chromatin such as euchromatin and the heterochromatin. The euchromatin involves highly active DNA required for the transcription process. It stains lighter than the heterochromatin. Males possess X and Y chromosomes. The females possess two X chromosomes. The sizes and the shapes of the chromosomes vary as per the phase of the cell cycle. The size of the metaphase chromosome is 5mm. Chromosomes acquire different shapes during the anaphase such as the rods, V, J, T, or X shaped.

A metaphase chromosome reveals five main structural components such as the satellite, the telomere, the chromatids, the primary constriction, and the secondary constriction. The two symmetrical halves running parallel to one another indicate the chromatids. The ones adjacent to each other are known as sister chromatids. The primary constriction is also known as the centromere. It pins up the chromatids together. The centromere divides the chromosome into its respective arms. The short chromosomal arm is known as the p arm. The long chromosomal arm is known as the q arm. The secondary constriction or the nucleolar organizer region gets associated with the nucleolus and its formation. The position of the centromere is different for every chromosome.

Chromosome pair number	Group of the chromosome	Type of the chromosome based on its structure	Abnormalities associated with the structure or number
1	A	Metacentric	1p36 deletion syndrome
2	A	Submetacentric	2q37 deletion syndrome Cancers
3	A	Metacentric	3p deletion syndromes Microdeletion syndromes Cancers
4	B	Submetacentric	Cancers Wolf-Hirschhorn syndrome
5	B	Submetacentric	5q31.3 microdeletion syndrome  Cri-du-chat syndrome
6	C	Submetacentric	6q24-related transient neonatal diabetes mellitus  Cancers
7	C	Submetacentric	Russell-Silver syndrome Saethre-Chotzen syndrome Williams syndrome
8	C	Submetacentric	Recombinant 8 syndrome Trichorhinophalangeal syndrome type II
9	C	Submetacentric	Bladder cancer Chronic myeloid leukemia Kleefstra syndrome
10	C	Submetacentric	Cancers
11	C	Submetacentric	Jacobsen syndrome Neuroblastoma
12	C	Submetacentric	Pallister-Killian mosaic syndrome
13	D	Acrocentric	Retinoblastoma Trisomy 13
14	D	Acrocentric	FOXG1 syndrome Multiple myelomas Ring chromosome 14 syndrome
15	D	Acrocentric	Prader-Willi syndrome Angelman syndrome
16	E	Metacentric	16p11.2 deletion syndrome 16p11.2 duplication Rubinstein-Taybi syndrome
17	E	Submetacentric	17q12 deletion syndrome 17q12 duplication Acute promyelocytic leukemia Miller-Dieker syndrome Potocki-Lupski syndrome Smith-Magenis syndrome
18	E	Submetacentric	Tetrasomy 18p Trisomy 18
19	F	Metacentric	19p13.13 deletion syndrome
20	F	Metacentric	Alagille syndrome Cancers Ring chromosome 20 syndrome
21	G	Acrocentric	Down’s syndrome
22	G	Acrocentric	22q11.2 deletion syndrome 22q11.2 duplication 22q13.3 deletion syndrome Emanuel syndrome
X	-	Submetacentric	Klinefelter syndrome Triple X syndrome Turner syndrome X-linked acro gigantism
Y	-	Acrocentric	47, XYY syndrome 48, XXYY syndrome Y chromosome infertility

Karyotyping:

It helps in grouping the chromosomes from A to G. Human cells possess 22 pairs of autosomes (non-sex chromosomes). There are two sex chromosomes known as X and Y respectively. The procedure of karyotyping involves three main steps such as culturing the cells to get the chromosomes, banding or staining technique, and observation under the microscope followed by software analysis. After arranging the chromosomes in respective groups, they get analyzed for the presence or absence of the structural or numerical variations.

Image 2: Chromosome Banding

Chromosome banding:

The banding techniques help in identifying the dark and the light regions or chromosome bands. Various techniques exist in chromosome banding. The G-banding is the most commonly used technique. It involves treating the chromosomes with trypsin. The trypsin denatures the proteins present in the chromosome. The next step involves staining the chromosomes with Giemsa solution. The light and dark bands form due to this type of staining technique. Thus, it is possible to visualize them under the microscope. The Q banding method helps in staining the chromosomes with quinacrine mustard. The banding patterns mimic that of the G banding. The R-banding technique involves pre-heating of the chromosomes before staining with the Giemsa. It involves a reverse banding pattern as compared to the G banding. The Centromere and the secondary constriction regions get stained using C- banding.

Fluorescence in-situ hybridization:

The FISH technique utilizes a single-stranded DNA probe labeled with fluorescent labels. The single-stranded DNA probe gets annealed with the complementary target sequence on the chromosome. Hence, it detects the regions with similar sequences. Three main types of FISH probes include Centromeric probes, chromosome-specific unique sequence probes, and whole chromosome paint probes. The sequences found near the Centromeric regions show the presence of repetitive DNA. Also, the regions of centromere themselves show a large number of repetitive sequences. Thus, Centromeric probes help in identifying the sequences. The sub-microscopic deletions and duplications get identified using the chromosome-specific unique sequence probe. To visualize the entire chromosome, the cytogeneticists use whole chromosome paint probe. 

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Medical Genetics

Genetics plays an important role in the field of medical sciences. There arises a genetic base to a disease, the development, reproduction, and other processes in the body. The gene or a blueprint of life occurs in every cell and is an important inheriting factor. Hence, it is important to understand genes and their role in various diseases, cancers, developmental stages, and interactions with various components. Some of the genes encode for proteins. The genes get passed on from one generation to the next generation. They also carry any minute changes or mutations. The diverse population is a sign of variation. Due to recent advances in health research, more and more diseases showed a genetic base. The science of genetics helps in detecting many diseases. It helps in their treatment or prevention. When you try to understand the molecular intricacies of the genetic material, you will be amazed to see an entirely different world. Thousands of reactions keep occurring at a molecular level.

DNA, the blueprint of life consists of nucleotide base pairs attached to the phosphodiester bonds. Small units of DNA form genes and exhibit specific functions. Various disorders arise due to gene mutations. Hence, they are known as genetic disorders. Detection of genetic disorders involves modern genetic diagnostic techniques. There involves the tremendous importance of genetics in medical sciences. To understand its role in health and disease, we must know the basic concept of genetics. 

The review covers the following topics in detail:

1.     What are genes and various branches of genetics?

2.     Chromosomes and disorders associated with the genes

3.     Disorders inherited due to faulty genes

4.     The role of gene mutations in biochemical disorders

5.     Detection of genetic disorders

6.     Genetic counseling

7.     Gene Cloning and DNA analysis

8.     Gene therapy

Image 1: Medical genetics

Various branches of genetics:

Human genetics involves several branches such as cytogenetics, molecular genetics, biochemical genetics, cancer genetics, Immunogenetics, and developmental genetics. Cytogenetics is the study of chromosomes and various disorders associated with the same. A chromosome is a specialized structure consisting of DNA-protein complex packed in a condensed manner. Cytogenetic techniques help in studying these structures. Molecular genetics involves the study of genes at the molecular levels such as single base pair changes, mutations, and other studies. Biochemical genetics involves genes controlling the enzyme production. Cancer genetics involves studying cancer genes and mutations associated with cell cycle control. Different antigen-antibody interactions studies involve immunogenetics. Developmental genetics helps to study the genetic control of the development. Hence there are many such branches of genetics. The field of genetic science is very vast. Half the rate of first trimester abortions involves chromosomal abnormalities. Congenital malformations, childhood blindness, deafness, and mental retardation mostly occur due to gene mutations.

Chromosomes and disorders associated with the same:

Every cell in the human body constitutes chromosomes. The chromosome complement in humans consists of autosomes and sex chromosomes. There are 22 pairs of autosomes and a pair of sex chromosomes. They are known as X and Y chromosomes respectively. A normal male has 22 pairs of autosomes, an X, and a Y chromosome. A normal female has 22 pairs of autosomes and two X chromosomes. Karyotyping involves arranging the chromosomes as per the groups resulting in a photomicrograph. This photomicrograph or a karyotype consists of autosomes and sex chromosomes arranged in groups. Structural and numerical abnormalities in the chromosomes result in genetic disorders. They are known as chromosomal anomalies or chromosomal abnormalities.

Disorders due to an abnormal number of chromosomes are known as numerical anomalies. They arise due to non-disjunction of chromosomes. Monosomies (one chromosome less), trisomies (one chromosome extra), and many other conditions consist of chromosomal anomalies. Down’s syndrome is an example of trisomy. It occurs due to trisomy of the 21^st chromosome. Disorders associated with the structural abnormalities of chromosomes are known as structural anomalies. Deletions, duplications, inversions, and translocations arise due to the structural anomalies of the chromosomes. They also result in conditions such as mosaicism.

Disorders inherited due to faulty genes:

Monogenic or single gene disorders are the genetic disorders arising due to the inheritance of a mutated gene. Two main types of inheritance include autosomal inheritance and sex-linked inheritance. Autosomal inheritance involves autosome related disorders or traits. Inheritance of the traits due to the expression of genes present on the sex chromosomes is known as a sex-linked inheritance. Autosomal dominant inheritance manifests the trait even if the mutant gene occurs in a single dose. For example, Huntington’s disease arises due to abnormal CAG nucleotide repeat. Autosomal recessive inheritance manifests the trait even if the gene is present in the double dose (homozygous). An example includes sickle-cell anemia. Sex-linked inheritance is either X-linked or Y-linked. The X-linked inheritance occurs in a dominant or a recessive form. Very few cases report Y-linked inheritance.

Polygenic or multifactorial inheritance depends on many genes. The traits are known as quantitative traits and depend on many factors. Abnormalities in mitochondrial DNA (mtDNA) occur due to mitochondrial inheritance. The genes do not behave as dominant or recessive in polygenic inheritance. They exhibit an additive effect on the trait.

Biochemical genetics:

This area of genetics deals with the genetic control of the metabolic pathways. According to the one gene-one enzyme hypothesis proposed by Beadle and Tatum, metabolic processes occur in various steps controlled by enzymes. Each enzyme gets coded by one gene. Inborn error of metabolism arises due to enzyme unavailability or insufficiency occurring as a result of related gene mutations. The inborn error of metabolism follows Mendelian inheritance pattern. PKU, a classic example of the inborn error of metabolism, arises due to the deficiency of phenylalanine hydroxylase.

Image 2: The role of genetics in medicine

Detection of genetic disorders:

Cytogenetic and molecular genetic studies or tests detect various anomalies. The prenatal diagnosis helps to detect abnormalities in the fetus before birth. Examples of prenatal tests include Amniocentesis, chorionic villus sampling, fetoscopy, ultrasonography, maternal serum screening, and fetal blood sampling. Non-invasive prenatal tests also help in detecting the fetal DNA through maternal serum testing. Karyotyping helps to detect chromosomal abnormalities. FISH and other hybridization techniques detect minute changes in the DNA. SNP genotyping involves PCR, agarose gel electrophoresis, and gel documentation systems.

Genetic counseling:

It is a specialized session with a genetic counselor. The genetic counselor is an expert in genetics and a trained professional who guides the couples and patients suspected with the genetic disorders. Genetic counseling helps in the risk assessment of hereditary diseases, repeated abortions, and stillbirth cases. It also helps in reducing the risk of having a baby with the genetic and other ailments including the inborn errors of metabolism.

Gene cloning and DNA analysis:

Gene cloning involves cloning the gene of interest for incorporation into the desired vector. It has a wide range of applications in recombinant DNA technology. It helps in expressing the desired products such as proteins, vitamins, and other molecules. Peptide vaccines and hormones get developed using gene cloning. DNA analysis using techniques such as DNA fingerprinting help to solve parental issues. Analyzing DNA and genotyping techniques help in detecting gene mutations.

Gene therapy:

It helps in replacing the gene that has lost its function. The first step involves defective gene identification and cloning the normal gene in place of the defective gene. The normal gene insertion includes a vector. The first gene therapy gained success in case of a child having ADA deficiency associated with severe combined immunodeficiency syndrome (SCID). 

References:

[1] Emery’s elements of Medical Genetics, Peter D. Turnpenny

[2] Chromosome Abnormalities and Genetic Counseling,  R.J. MKinlay Gardner, Grant R Sutherland, Lisa G. Shaffer
[3] Medical Genetics, G. Bradley Schaefer, James N. Thompson

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