Brief notes on Griffith’s Transformation Experiment

In 1928 F. Griffith made a series of unexpected observations while experimenting with a pathogenic (disease causing) bacterium, Diplococcus pneumoniae (then known as Pneumococcus).

This bacterium causes pneumonia in man and most mammals and has two phenotypes. One is the virulent/ pathogenic form and possesses a polysaccharide coat that protects the bacterium from phagocytic attack of the host. Because of the coat the virulent bacteria form smooth edged colonies in culture.

The other is avirulent/ non-pathogenic and lack the coat. They form rough edged colonies in cultures. The virulent forms are, therefore called Smooth-type or S- type and the avirulent forms are called Rough-type or R-type.

There can be several strains of S- type and R-type, like S-I, S-II, S-III and R-I, R-II, R-III etc. Griffith selected mouse as the host and S-III and R-II bacteria for his experiment. It was apparent that mouse injected with S-III bacteria suffered from the disease and died while those injected with R-II did not suffer and survived. Bui Griffith made some surprising observations when he injected mice with different combinations of bacteria. His experimental findings can be summarized as:

Mouse injected with live S-III Died

Mouse injected with live R-II Survived

Mouse injected with

Heat killed S -III Survived

Mouse injected with

Heat killed S -III + live R-II Died.

Extract from dead mouse | (cultured) Live R-II+Live S-III

This indicated that live S-III extracted from the dead mouse initially injected with heat killed S-III and live R-II must have arose from R-II. It could not have been due to mutation in R-II; in that case live S-II not live S-III would have been formed.

The dead S-III and live R-II would have interacted in some way so that some of the live R-II would have been transformed to live S – III.From the heat killed cells of S-III “something” would have escaped and transformed R-II to S- III. This “something: was referred to as transforming principle. Griffith was unaware of the nature of the transforming principle.

Subsequent proof for the chemical nature of Griffith’s transforming principle was provided by Oswald T. Avery and his co-workers Maclyn McCarty and Colin M. Macleod of Rockfcllcr Institue, New York, U.S.A. in 1944.

They performed in vitro experiments with highly purified DXA extract of heat killed S-III bacterium. They used the extracted DXA along with combinations of different enzymes to transform the R-II type bacteria.

This DXA extract retained its transforming ability when subjected to protease (that digests protein) or Ribonuclease (that digests RXA), but lost the transforming ability when subjected to Deoxyribonuclease (that digests DXA). This experiment can be summarized as below:

R-II +DXA extract of S-III + no enzyme = R-II colonics + S-III colonies

R-II + DXA extract of S-III +Ribonuclcase = R-II colonies + S-III colonies

R-II + DXA extract of S-III + Protease = R-II colonics + S-III colonies

R-II + DXA extract of S-III + Deoxyribonuclease = Only R-II colonics.

This experiment showed that when the preparation was treated with DXA digesting enzyme Deoxyribonuclease, no transformation of R-II strains to S-III strains occurred. This provided the first evidence for DXA as the transforming principle or the genetic material.

Alfred D. Hershey and Martha J. Chase in 1952 provided the direct evidence for DXA as the infecting principle and the genetic material in T2 bacteriophage. It was conclusively proved that the DXA of the chromosome not the protein is the genetic material. Further works also proved that in some viruses RXA is the genetic material. The first evidence in support of RXA as the genetic material was provided by Conrat and Singer in tobacco mosaic virus.

Early geneticist by now had recognized certain basic properties of gene, like:

* That gene is a chromosomal site controlling observable characteristics.

* That gene can be mutated or changed.

* That gene can rccombine with homologous site on another chromosome.

* That gene can replicate and transmit to next generation.

Once the chemical nature and the basic properties of genetic materials were established then the question remained to be answered were how the genes control characters and how they transmit characters to the next generation. In 1941, George Beadle and Edward Tatum of Stanford University through a series of experiment provided the answer to how genes control characters.

They deliberately set Mendelian mutations in bread mold Neurospora and studied their effects. From this they concluded that one gene is responsible for the synthesis of one enzyme: “one gene one enzyme hypothesis”. Later on this concept was modified to “One gene one polypeptide” hypothesis as many enzymes or proteins consist of multiple protein or polypeptide subunits.

This was the first clear statement about the relation between the genotype and phenotype. Once it was established that a gene is responsible for a polypeptide then the question was how a gene determines a particular polypeptide.

Iⁿ J-953 Watson and Crick unraveled the double helical structure of DXA. In the same year English biochemist Frederick Sanger announced the complete sequence of amino acids of Insulin protein.

This work of Sanger proved that protein consist of defined sequence of amino acids. Watson and Crick model also proved that DNA consists of defined sequence of nucleotides. Then just a little more insight could provide the relation between the defined nucleotide sequence of DXA (genetic material) and defined amino acid sequence of protein.

The general understanding of the co linearity of nucleotide sequence of genetic material and amino acid sequence of protein came from the study of Sickle cell anemia by Vernon Ingram in 1956. In sickle cell anemia, the a-chain of hemoglobin contains amino acid valine at the sixth position instead of glutamine found in normal hemoglobin. This causes wrong folding of a-chain altering the tertiaiy structure of hemoglobin and reduces its capability to carry oxygen.

The alleles of the genes coding these two different polypeptide differ only in one nucleotide. His studies as well as other experimental evidences established that the nucleotide sequence of a gene determines the amino acid sequence of a protein. In other words a particular gene is responsible for the synthesis of a particular protein or polypeptide.

In 1955 Benzer showed that the units of genetic material that can mutate, that can recombine or that can determine the synthesis of a polypeptide were different. According to him the smallest part of DXA that can undergo mutation is muton.

The smallest unit of DXA capable of recombination is recon and the smallest unit capable of polypeptide synthesis is cistron. Cistron is much larger than recon and muton is the smallest. A gene can be monocistronic or polycistronic.

The Genes: Chemically genes arc DXA or rarely RXA.It is evident that genes have fixed positions on chromosomes and each gene has a defined boundary. A gene can be defined as a sequence of DXA responsible for the coding of a functional polypeptide or rRXA, tRXA and other forms of RXAs.

A typical eukaryotic gene contains a structural region coding for RXAs and a promoter or controlling region regulating gene expression. The promoter varies in length in different genes and is usually located upstream of structural region (towards 5′ side). It can be several hundred nucleotides long.

The positions of upstream nucleotides are designated with minus (-) sign and those of downstream are designated with plus (-t-) sign. Some sequences in promoter are conserved (similar not identical) in many genes such as:

TATA box; It is a six to eight base pair A and T rich sequence, located at -20 or -25 base pair upstream.

G-C box: It is a G and C rich region of Six to eight base pair located at -50 positions.

CAAT box is at -75 positions and again at -100 or -110 position there is another G-C box Enhanccr clement

Enhancer elements are located much farther upstream; about some kilo base pairs away.

All these promoter elements may not be there in all genes. These elements or DXA sequences are concearned with regulating the expression of genes. The promoter is responsible for binding of RXA polymerase enzyme (described later) and transcription initiation factors (TIF).

The structural region in most eukaryotic genes contains two distinct types of regions: the exons and the introns. The exons are the expressing regions responsible for the coding of a functional polypeptide.

Introns or intervening sequences (IVS) or spacer sequences are not expressed or they don’t code for any type of RXA. Such genes are known as interrupted or split genes. Richard Roberts and Phillip Sharp independently discovered split gene in 1977. Both of them were awarded with Xobel Prize in 1993. Some genes in eukaryotes do not have introns, like the genes coding for histones and interferons.

These genes having no introns are known as exonic genes. Some prokaryotic genes like thymidylatc synthetase gene and ribonucleotide reductase gene in T4 phage are split genes. In length the introns are much larger than exons.

The average length of introns is about 3,500 nucleotides and that of exon is 150 nucleotides.The human muscle protein titin is the largest single chain protein (26,926 amino acid) and its gene has the highest number of introns (234).

The prokaryotic gene also has promoter and structural region. The promoter is about 40 base pair long with two conserved or consensus sequences. In E.coli two important consensus sequences
arc at -10 and -35 positions. The -10 position is a hexanuclcotidc of TATAAT known as Pribnow box, after David Pribnow who described it. The -35 sequence has TTGACA. The nucleotide from which the transcription starts is +1 and usually is A or G. The structural region in prokaryotcs usually has no introns.