•  Scientific Works

Prof. S. Riazuddin started research career in 1964 with M.Sc. thesis work on "the nutritive value of leaf protein concentrates". These studies showed that a six hour incubation of defatted leaf protein concentrates at 60 o C, improved the shelf life of the product without affecting its digestibility when studied by in vitro protease hydrolysis, using pepsin, trypsin or pancreatic extracts. In 1967, he started to work for Ph.D. thesis on calcium metabolism. These studies revealed that elevated mineral requirements in ewes during such physiological stress conditions as pregnancy – parturition – lactation, were met largely by exchangeable calcium of the skeleton; and depleted mineral skeletal stores of the mother were made good by a gradual increase in absorption of dietary calcium when physiological stresses disappeared. Further, ageing of the animal and subcutaneous implantation of steroid hormones improved calcium retention levels as well as sizes of the exchangeable calcium pools.

In 1969-70, two Nobel prize discoveries -- that of a site-specific endonuclease in H.O. Smith's laboratory and a reverse transcriptase by Howard Temin & David Baltimore aroused his interest in molecular biology. In 1970, he started post-doctoral research in Professor Bryn Bridges laboratory at the University of Sussex and continued this work in Pakistan (1971-73). These studies demonstrated that transfer of donor DNA to the recipient cells, during bacterial conjugation, was unaffected when donor cells were irradiated with UV/gamma radiations. It was further observed that damaged DNA base residues transferred from irradiated donor to un-irradiated recipient cells during bacterial mating, were faithfully corrected in repair proficient recipient cells.

Fascinated by the intricacies of cellular repair processes, he joined Professor L. Grossman's laboratory to investigate the molecular mechanism of DNA repair in Micrococcus luteus . The choice to employ Micrococcus luteus as a model bacterial system was based mainly on a) its unusual UV resistance thus offering a good bacterial source of UV repair enzymes, b) extreme sensitivity to alkylating chemicals thus providing an ideal cellular system to induce chemical resistance; and c) low levels of endogenous non-specific endonucleases.

Ultraviolet irradiation of cellular DNA produces mainly cyclobutane type prymidine-prymidine dimers and a small proportion (1.5%) of 5,6 dihydroxy dihydrothymine glycol modifications. The lethal and mutagenic effects of UV radiation can be attributed to inefficient repair of thymine dimers in DNA. Alkylating chemicals interact with cellular DNA to produce at least 10 different base modifications having different physiological effects. SN1 type alkylating chemicals such as dimethyl sulphate (DMS) and methylmethane sulphonate (MMS) modify ring nitrogens in the purines. On the other hand SN2 type alkylating chemicals such as N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and N-methyl-N-nitrosourea (MNU) high reactivity towards oxygen atoms in DNA. Exocyclic oxygens in DNA bases and internucleotide phosphate groups are a common site of attack, yielding a variety of alkylation products which have different physiological effects. The presence of O 6 -methylguanine, O 4 -methylthymine or O 2 -methylthymine has been implicated in the mutagenic/carcinogenic effects of alkylating chemicals. Most of the studies on alkylation damage and repair had focussed attention on the physiological fate of ring nitrogen modifications which constitute about 60-95% of total alkylation products. The fate of highly carcinogenic exocyclic oxygen modifications had been ignored partly because of difficulties in their detection and partly due to inducible nature of proteins involved in the repair of minor alkylation products.

Dr. S. Riazuddin's work on DNA repair in M. luteus has made significant contributions to the understanding of molecular processes leading to eventual repair of physiologically important base modifications. It has been established that repair of prymidine-prymidine dimers is initiated by a specific UV endonuclease- glycosylase enzyme with the introduction of a cleavage at the N-glycosylic linkage. This enzymatic cleavage terminates in a base-free deoxyribose originally associated with the '3 thymine residue of the dimer and 5' phosphorylated nucleotide. The resulting apurinic site is removed by cellular apurinic endonucleases. On the other hand, repair of thymine glycol produced as a minor UV product, is initiated by the introduction of an endonucleolytic incision on the 5' side of the damaged base residue by a distinctly separate damage specific endonuclease. The modified base is removed by the exonucleolytic function of DNA polymerase I. Continuity of the repaired strand is restored in either case by the combined functions of DNA polymerase I and polynucleotide ligase.

Contrary to the mode of UV repair, cellular repair of alkylated DNA base modifications in M. luteus depends primarily upon the previous exposure of cells to alkylating chemicals. Pretreatment of cells with sublethal concentrations of N-methyl-N-nitro-N-nitrosoguanidine induces 5.7 fold increased resistance to the lethal effects of challenge doses of the same alkylating chemical. This phenomenon termed as adaptive response is accompanied by the synthesis of at least eight proteins. Five of these proteins are DNA glycosylase enzymes numerically designated as GI-GV in order of their decreasing molecular weights. GI, is a constitutive heat labile protein which acts exclusively on 3-methyladenine base residues in alkylated DNA. GII, which is an inducible protein, acts on 3-methyladenine, 3-methylguanine, 7-methyladenine, and 7-methylguanine with unequal facility. GIII, is another inducible protein which acts on O 2 -methylthymine and O 2 -methylcytosine with no activity on methylated purines. The identity of GIV & GV proteins has yet to be established. The remaining three proteins, identified as methyl transferases are designated as TI, TII, TIII enzymes in order of their elution from a Sephadex G75 column. All three methyl transferases have very narrow substrate specificities with TI acting exclusively on O6-methylguanine base modifications; T2 acting only on O4-methylthymine and T3 showing absolute specificity for stereo specific phosphotriesters. All of these enzymes have been highly purified, biochemically characterized and their substrates specificities established. Cellular role of the isolated repair proteins is being investigated by identifying structural genes of these proteins and their cloning on multicopy plasmid DNA in E.coli .

Recognizing the absolute need for a reliable and continuous supply of Type-II restriction enzymes in genetic engineering and recombinant DNA work in Pakistan , Professor S. Riazuddin initiated a programme to create a local repository of common molecular tools, and simultaneously searched for new Type-II specificity restriction enzymes. As part of a collaborative effort, involving Dr. R.J. Roberts (Nobel Laureate) and his group at Cold Spring Harbor Laboratory, N.Y., USA , his laboratory has discovered Forty-five new restriction enzymes present in different microbes. The discovery of these enzymes have greatly enhanced the powers of genetic manipulations through recombinant DNA methologies. All the new enzymes have been entered in International DataBank and two of these enzymes ( Bse RI and Bsc AI) are being marketed by New England Biolabs, Boston , USA .

Dr. S. Riazuddin has searched systematically the Pakistan environments for new and novel isolates of Bt possessing pesticidal properties against rice stem borers, rice leaf folder, cotton pink ball worm, chickpea army worm, coleopteral tribolium & rape-seed aphids, and established a local repository containing over 500 isolates varying in genetic make-up and showing different insecticidal properties. Using local isolates as the source material, cryIA(a), cryIA(b), cryIA(c), cryIII and cryIV genes (classification) have been cloned and further manipulated to optimize expression levels in plants. This provides a precious genetic resource for genetic engineering of plants for breeding insect resistance.

Dr. S. Riazuddin has discovered two novel pesticidal genes which have larvicidal properties against agronomically important pest such as nematode, aphids and white fly. Bacillus thuringiensis parasporal proteins have been previously reported to have insecticidal properties against lepidopteral and dipteral insects. Dr. Riazuddin has for the first time, unequivocally demonstrated that pesticidal activity of Bacillus thuringiensis parasporal protein is not limited to lepidopteral and dipteral insects, but the activity spectrum extends to other agronomically important pests. This has opened new avenues of research and is considered a scientifically important discovery. The excitement may be judged from the fact that a number of laboratories in Europe, USA and Australia are offering to collaborate with Dr.Riazuddin in further exploiting the larvicidal potential of the said novel isolates which are being patented in USA, in a joint venture with Calgene, Inc., Davis, California, USA.

In 1983, Dr. S. Riazuddin attended a 6-week plant molecular biology course organized by Professor F. Ausubel ( Harvard University ) at Cold Spring Harbor Labs, N.Y., USA . His participation in the course introduced him to the revolutionary potential of plant molecular biology. In 1988, he spent a sabbatical year in Eugene Nester/M.P. Gordon's laboratory in the University of Washington , Seattle , USA to learn Agrobacterium mediated plant transformation. Recognizing the obvious role of plant biotechnology in the future economic growth and development of Pakistan , he initiated a solid and definitive programme in plant molecular biology to exploit transgenic plant technology for breeding genetic resistance against local pests/fungi in rice, cotton and chickpea.

Dr. S. Riazuddin's laboratory has genetically transformed cotton (MNH-93) using Agrobacterium-mediated transformation system with bacterial pesticidal genes from Bacillus thuringiensis [cryIA(b)]. Genetically transformed plants show inherent resistance against American bollworm (Heliothis armigera). To genetically engineer monocotyledonous plants, Dr. Riazuddin has locally developed a particle acceleration gun (biolistic device) at 1/20th the cost of a proprietary device marketed by M/s Dupont , USA through BioRad. The locally developed device has been successfully used to transform indica rice Basmati 370 with Bt pesticidal genes [cryIA(b), cryIA(c) & CryIIA]. Transgenic Basmati 370 rice plants exhibit inherent resistance against rice leaf-folder and rice stem-borer.

Recognizing the revolutionary potential and keidoscopic openings stemming from the data/ information revealed from the sequencing of human genome and birth of associated DNA analytical techniques, Dr. Riazuddin has initiated a programme of studies to elucidate the molecular basis of human genetic diseases. Identification of human genes/pathogenic mutations causing b -thalassemia, and hearing and vision impairment in Pakistani population. As a result of input into this programme, ten new linkages for recessive deafness, fifty new pathogenic mutations, and four novel deafness causing gene have been identified in Pakistani consanguineous families. Further, three rare b -Thalassemia mutations (unreported so far in Asian population), one rare a -hemoglobin variant have been identified in Pakistani population. On the basis of results obtained in laboratory experimentation, prenatal diagnostic procedures have been developed which are being practiced in collaboration with local gynecologists to counsel families with b -thalassemic history. Further, Human DNA typing is being used for forensic.

•  Development Works

In 1981, Dr. S. Riazuddin was selected, a member of expert panel constituted by the United Nations Industrial Development Organization (UNIDO) to examine the implications of scientific and technological breakthroughs in genetic engineering and biotechnology in relation to the future economic growth of developing countries. The committee comprised scientists of exceptional repute including Professor R.H. Boyer (California, USA), S. Narang, (Ottawa, Canada), R. Wu, (Cornell, USA), A. Chakrabarty, (Chicago, USA), A. Bokhari, (New York, USA), Carl Heden (Stockholm Sweden) with Dr. S. Riazuddin, (Faisalabad, Pakistan; the only scientist from outside of the advanced world). In this capacity, Professor S. Riazuddin contributed to the preparation of a proposal for the establishment of an International Centre for Genetic Engineering and Biotechnology (UNIDO publication No.IS 254). In addition, Professor S. Riazuddin prepared a proposal for the establishment of Centres for Production of DNA Enzymes (UNIDO publication No.IS 271) and a paper on capability building in biotechnology and genetic engineering in developing countries (UNIDO publication No.IS 608).

In 1982, Professor S. Riazuddin was selected a member of the International Working Group of Experts in Chemical Carcinogenesis and Related Discipline to evaluate the carcinogenic risk of some food additives, feed additives, and naturally occurring substances. As member of the expert working group, he contributed to the preparation of Vol.31 of the IARC monographs on the Evaluation of Carcinogenic Risk of Chemicals to Humans.

In order to promote the applications of genetic engineering and biotechnology in the economic growth and development of Pakistan, and to build national capability in biotechnology, he conceived the establishment of a Centre for Advanced Molecular Biology jointly financed by the Federal Ministries of Education and Science & Technology with Dr. Riazuddin as its founding Director. Under his leadership, the Centre has emerged as one of the best places of teaching and research in molecular biology in this part of the world. The Centre is operating as a National Centre of excellence with four major functions.

•  To generate a cadre of manpower specifically trained in genetic engineering and recombinant DNA technology through regular M.Phil/ Ph.D. programmes as well as through short specialized training courses.

•  To undertake quality molecular biological research on problems selected on the basis of their impact on the understanding of mechanisms and processes which will lead to applications in health & medicine, agriculture, industry and energy sectors.

•  To create a repository of DNA enzymes, linker molecules, cloning vehicles, novel bacterial strains and other such molecular tools for ready availability and use by various research groups at this Centre and other research laboratories in Pakistan.

•  To organize national and international seminars and conferences for in-depth discussions on scientific and technological developments, leading to new ideas and innovative applications of knowledge on gene cloning and recombinant DNA to solve unique and specific problems in agriculture, health, energy and industrial sectors.

The Centre is advised by a committee of eminent scientists whose discoveries have led to the development of the new bioscience. Members of the committee include Prof. H.O. Smith (Nobel Laureate), Prof. R.J. Roberts (Nobel Laureate), Prof. L. Grossman (Distinguished Service Professor), V.C. Knauf (Vice-President Calgene), Professor Marc Van Montagu (Ex-President Plant Genetic System), Professor L. Loeb (Ex-President American Cancer Society).

Dr. S. Riazuddin's efforts have enabled to establish academic ties with some of the best institutions in Europe and USA . Such linkages have greatly contributed to the growth of his laboratory into a Centre of Excellence for research and teaching in molecular biology in this part of the world. Further, such arrangements have helped to overcome inherent difficulties in promoting the uses of DNA technology, and in creating a cadre of specifically trained manpower in Pakistan and in neighbouring developing countries.

Dr. Riazuddin has made significant contribution in the development of biotechnology capability of many developing countries. He has helped Bangladesh and Tunisia in the establishment of nucleus laboratories and organized training courses to generate a cadre of specifically trained manpower. In collaboration with COMSTECH and IFSTAD, he has helped to establish training courses, group meetings, symposia and seminars in Indonesia , Malaysia , Saudi Arabia and Egypt . Such activities have helped to create awareness about the unlimited economic potential of the new science of molecular biology and attendant technologies.

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